Reagents and methods for smooth muscle therapies

ABSTRACT

The present invention provides novel polypeptides comprising heat shock protein 20 (HSP20)-derived polypeptides to treat or inhibit smooth muscle vasospasm, as well to treat and inhibit smooth muscle cell proliferation and migration.

CROSS REFERENCE

This application claims priority from U.S. Provisional PatentApplication Ser. No. 60/314,535 filed Aug. 23, 2001, the disclosure ofwhich is incorporated by reference herein in its entirety.

STATEMENT OF GOVERNMENT FUNDING

The U.S. Government through the National Institute of Health, providedfinancial assistance for this project under Grant No. RO1 HL58027-06.Therefore, the United States Government may own certain rights to thisinvention.

FIELD OF INVENTION

This invention relates generally to the fields of cell biology,molecular biology, pharmaceuticals, and smooth muscle biology.

BACKGROUND

There are three types of muscles: cardiac, skeletal, and smooth. Smoothmuscles are found in the walls of blood vessels, airways, thegastrointestinal tract, and the genitourinary tract. The caliber oftubes lined by these muscles is dependent on a dynamic balance betweenthe state of contraction and the state of relaxation of the muscles inthese organs. Contraction and relaxation of smooth muscles are mediatedby different signaling pathways inside the muscles. Pathways whichinduce relaxation also inhibit contraction. Sustained contraction ofmuscle is a “spasm” of the muscle. This spasm can be prevented byactivating pathways or systems which induce relaxation, or in otherwords, inhibit contraction.

For the most part, smooth muscles are unique in that they lack theordered structure of cardiac and skeletal muscles and that they are ableto maintain tonic contractions with minimal oxygen use. Pathologic toniccontraction is a state in which the muscles are in spasm.

Many pathological conditions are associated with spasm of vascularsmooth muscle (“vasospasm”), the smooth muscle that lines blood vessels.Vasospasm, of the vessel causes narrowing of the vessel lumen, limitingblood flow. Spasm of any vessel leads to ischemia to the organ that thevessel supplies blood to. Ischemia is reversible lack of blood flow andoxygen supply to the tissues. In the case of spasm of the vessels in theheart it leads to cardiac ischemia and/or infarction; spasm of vesselsin the brain leads to stroke; spasm of the vessels that supply theintestines leads to mesenteric ischemia, a lack of relaxation of thevessels in the penis leads to impotence, since erection requiresvasodilation of the corpra cavernosal (penile) blood vessels; and spasmof the intracranial blood vessels leads to migraines.

Excessive vasoconstriction (or inadequate vasodilation) occur in otherdisease states as well. Hypertension (high blood pressure) is caused byexcessive vasoconstriction, as well as thickening, of the vessel wall,particularly in the smaller vessels of the circulation. This process mayaffect the lung vessels as well and cause pulmonary (lung) hypertensionand asthma (bronchospasm). Other disorders known to be associated withexcessive constriction, or inadequate dilation of smooth muscles includetoxemia of pregnancy, pre-term labor, pre-eclampsia/eclampsia, Raynaud'sdisease or phenomenon, anal fissure, achalasia, hemolytic-uremia, andPrinzmetal's angina, a form of coronary spasm that causes angina. Spasmin the coronary arteries also occurs during mechanical manipulation ofcoronary arteries, such as during angioplasty and stenting. This spasmcan lead to ischemia and infarction.

Surgical procedures involving the vasculature are also complicated byvasospasm of smooth muscle, which may result in both short term and longterm complications including restenosis and vascular occlusion. There isa general pattern in which vasospasm, if persistent, leads toconstrictive remodeling/intimal hyperplasia, and ultimately vascularocclusion. Corrective surgical procedures, such as stenting of a bloodvessel, angioplasty, and implanting prosthetic devices such as dialysisaccess fistulas and shunts, are accompanied by damage to the smoothmuscle. This leads to smooth muscle cell proliferation and migration.This ultimately leads to constrictive remodeling and intimalhyperplasia. This process leads to restenosis, prosthetic graft failure,stent and stent graft failure, microvascular graft failure,atherosclerosis, and transplant vasculopathy.

While incompletely understood, intimal hyperplasia is mediated by asequence of events that include endothelial cell injury and vascularsmooth muscle proliferation and migration from the media to the intima.This is associated with a phenotypic modulation of the smooth musclecells from a contractile to a synthetic phenotype. The “synthetic”smooth muscle cells secrete extracellular matrix proteins, which leadsto pathologic vascular occlusion, as described above. Furthermore,increased proliferation and migration of smooth muscle cells can alsolead to smooth muscle cell tumors, such as leiomyosarcomas andleiomyomas.

Thus, it would be of great benefit to identify new methods andtherapeutics to treat or inhibit smooth muscle vasospasm, promote smoothmuscle relaxation, improve other therapies involving smooth muscle, andto treat and inhibit smooth muscle cell proliferation and migration.

SUMMARY OF THE INVENTION

The present invention provides new methods and therapeutics to treat orinhibit smooth muscle vasospasm, promote smooth muscle relaxation,improve other therapies involving smooth muscle, and to treat andinhibit smooth muscle cell proliferation and migration.

In one aspect, the present invention provides polypeptides consisting ofan amino acid sequence according to general formula I:X1-X2-[X3-A(X4)APLP-X5-]_(u)-X6

wherein X1 is absent or is one or more molecules comprising one or morearomatic ring;

X2 is absent or comprises a transduction domain;

X3 is 0, 1, 2, 3, or 4 amino acids of the sequence WLRR (SEQ ID NO:1);

X4 is selected from the group consisting of S, T, Y, D, E,hydroxylysine, hydroxyproline, phosphoserine analogs and phosphotyrosineanalogs;

X5 is 0, 1, 2, or 3 amino acids of a sequence of genus Z1-Z2-Z3,

wherein Z1 is selected from the group consisting of G and D;

Z2 is selected from the group consisting of L and K; and

Z3 is selected from the group consisting of S and T;

X6 is absent or comprises a transduction domain; and

wherein u is 1-5.

In a preferred embodiment, X4 is phosphorylated. In a further preferredembodiment, at least one of X2 and X6 comprises a transduction domain.

In another aspect, the invention provides polypeptides consisting of anamino acid sequence according to the general formula II:X1-X2-[X3-A(X4)APLP-X5]_(u)-X6wherein X1 is absent or is one or more molecules comprising one or morearomatic ring;

X2 is absent or comprises a cell transduction domain;

X3 is 0-14 amino acids of the sequence of heat shock protein 20 betweenresidues 1 and 14 of SEQ ID NO:297;

X4 is selected from the group consisting of S, T, Y, D, E,hydroxylysine, hydroxyproline, phosphoserine analogs and phosphotyrosineanalogs;

X5 is 0-140 amino acids of heat shock protein 20 between residues 21 and160 of SEQ ID NO:297;

X6 is absent or comprises a cell transduction domain; and

wherein at least one of X2 and X6 comprise a transduction domain.

In a preferred embodiment, X4 is phosphorylated.

In another aspect, the present invention provides pharmaceuticalcompositions, comprising one or more polypeptides of the presentinvention and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides isolated nucleic acidsequences encoding a polypeptide of the present invention. In furtheraspects, the present invention provides recombinant expression vectorscomprising the nucleic acid sequences of the present invention, and hostcells transfected with the recombinant expression vectors of the presentinvention.

In another aspect, the invention provides improved biomedical devices,wherein the biomedical devices comprise one or more polypeptides of thepresent invention disposed on or in the biomedical device. In variousembodiments, such biomedical devices include stents, grafts, shunts,stent grafts, angioplasty devices, balloon catheters, fistulas, and anyimplantable drug delivery device.

In another aspect, the invention provides methods for inhibiting smoothmuscle cell proliferation and/or migration, comprising contacting thesmooth muscle cells with an amount effective to inhibit smooth musclecell proliferation and/or migration of one or more polypeptide of thepresent invention. In various preferred embodiments of this aspect ofthe invention, the method is used to treat or prevent a disorderselected from the group consisting of intimal hyperplasia, stenosis,restenosis, and atherosclerosis. In various other preferred embodimentsof this aspect of the invention, the method is performed on a subjectwho has undergone, is undergoing, or will undergo a procedure selectedfrom the group consisting of angioplasty, vascular stent placement,endarterectomy, atherectomy, bypass surgery, vascular grafting, organtransplant, prosthetic implant, microvascular reconstructions, plasticsurgical flap reconstruction, and catheter emplacement. In a furtherembodiment of this aspect of the invention, the method is used to treatsmooth muscle cell tumors.

In a further aspect, the present invention comprises methods fortreating or inhibiting a disorder selected from the group consisting ofintimal hyperplasia, stenosis, restenosis, and/or atherosclerosis,comprising contacting a subject in need thereof with an amount effectiveto treat or inhibit intimal hyperplasia, stenosis, restenosis, and/oratherosclerosis of HSP20, or a functional equivalent thereof.

In a further aspect, the present invention comprises methods fortreating smooth muscle cell tumors comprising contacting a subject inneed thereof with an amount effective to treat smooth muscle tumors ofHSP20, or a functional equivalent thereof.

In a further aspect, the present invention provides a method fortreating or preventing smooth muscle spasm, comprising contacting asubject in need thereof with an amount effective to inhibit smoothmuscle spasm of one or more polypeptides of the present invention. Invarious preferred embodiments of this aspect of the invention, themuscle cell spasm is associated with a disorder or condition selectedfrom the group consisting of angina, Prinzmetal's angina (coronaryvasospasm), ischemia, stroke, bradycardia, hypertension, pulmonary(lung) hypertension, asthma (bronchospasm), toxemia of pregnancy,pre-term labor, pre-eclampsia/eclampsia, Raynaud's disease orphenomenon, hemolytic-uremia, non-occlusive mesenteric ischemia, analfissure, achalasia, impotence, migraine, ischemic muscle injuryassociated with smooth muscle spasm, and vasculopathy, such astransplant vasculopathy.

In a further aspect, the present invention provides methods forpromoting smooth muscle relaxation, comprising contacting smooth musclewith an amount effect effective to promote smooth muscle relaxation withone or more of the polypeptide of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Mesangial cells were transfected with vectors containing greenfluorescent protein (GFP) alone, GFP fused to the 5′ end of the wildtype cDNA for HSP20 (WT), or GFP fused to an HSP20 construct in whichthe PKA phosphorylation site was mutated to an alanine (S16A-HSP20)(MUT)and the number of wrinkles under the cells was determined aftertreatment with dibutyryl cAMP (10 ΦM) for 0 minutes, 30 minutes, 60minutes, or 90 minutes.

FIG. 2. Mesangial cells were transduced with FITC-TAT-HSP20 and thenumber of wrinkles under the cells was determined at the time pointsindicated using phase contrast microscopy (n=10, *=p<0.05 compared totime 0).

FIG. 3. Transverse strips of bovine carotid artery smooth muscle,denuded of endothelium, were pre-contracted with serotonin (1 μM for 10minutes), cumulative doses of FITC-phospho-HSP20-TAT, FITC-scrambledphosphoHSP20-TAT (FITC-NH₂-βAGGGGYGRKKRRQRRRPRKS*LWALGRPLA-COOH, opencircles) (SEQ ID NO:305), or FITC-TAT (FITC-NH₂-βAGGGGYGRKKRRQRRR,closed triangles) (SEQ ID NO:306) were added every 10 minutes, and thepercent contraction was calculated. The force is depicted as apercentage of the maximal serotonin contraction (n=5, *=p<0.05 comparedto 0 peptide added).

FIG. 4. Rings of porcine coronary artery in which the endothelium wasnot denuded, were pre-contracted with serotonin (1 μM for 10 minutes),cumulative doses of PTD-pHSP20 (NH₂-βAYARRAAARQARAWLRRAS*APLPGLK-COOH,closed circles) (SEQ ID NO:307) or PTD-scrambled-pHSP20(NH₂-βAYARRAAARQARAPRKS*LWALGRPLA-COOH open circles) (SEQ ID NO:308)were added every 10 minutes, and the percentage of relaxation wascalculated as a percentage of the maximal serotonin contraction (n=5,*=p<0.05 compared to 0 peptide added). The concentrations of peptideused are depicted on the x axis.

FIG. 5. Homogenates of mesangial cells (lane 1), rat aortic smoothmuscle cells (lane 2), and PKG transfected rat aortic smooth musclecells (lane 3) were immunoblotted for PKG (panel A) or HSP20 (panel B).In a separate experiment, mesangial cells were untreated (panel C) ortreated with dibutyryl cAMP (10 μM, 15 minutes, panel D). The proteinswere separated by 2-dimensional electrophoresis, transferred toimmobilon and probed with anti-HSP20 antibodies. Increases in thephosphorylation of HSP20 leads to a shift in the electrophoreticmobility of the protein to a more acidic isoform (arrow).

FIG. 6. Transfected mesangial cells were fixed, and the actin filamentswere stained with fluorescent-labeled phalloidin. Mesangial cells weretransfected with EGFP alone (EGFP), S16A-HSP20 (MUT-EGFP), or wild typeHSP20 (WT-EGFP). The cells were plated on a glass slides, and nottreated (CONT) or treated with dibutyryl cAMP (10 μM, for 30 minutes,db-cAMP). The cells were fixed and stained with rhodamine phalloidin.Dibutyryl cAMP led to a loss of central actin stress fibers in EGFP butnot S16A-HSP20 cells. In the cells overexpressing HSP20 the actin fiberswere peripherally localized.

FIG. 7. Bovine aortic endothelial cells were plated on glass coverslips(80K-100K cells) in DMEM plus 10% FBS over night (24 wells plate). Thecells were serum starved (no serum) for one hour and incubated in thepresence of the peptide analogues of HSP20(NH₂-βAYARRAAARQARAWLRRAS*APLPGLK-COOH-pHSP20 (10 uM) (SEQ ID NO:307) orscrambled analogues of HSP20(NH₂-βAYARRAAARQARAPRKS*LWALGRPLA-COOH-scHSP20 (10 uM)] (SEQ ID NO:308)for 30 minutes. The cells were fixed with 3% glutaraldehyde and thenumber of focal adhesions was detected with interference reflectionmicroscopy. The Hep I peptide was used as a positive control.

FIG. 8. Confluent A10 cells were serum starved (0.5% fetal bovine serum,FBS) for 48 hours. A linear wound was made in the smooth muscle cellmonolayer using a rubber scraper and the scratched edges were markedusing metal pins. The cells were changed to 10% FBS media containingPTD-pHSP20 (NH₂-βAYARRAAARQARAWLRRAS*APLPGLK-COOH (SEQ ID NO:307), orPTD-scrambled-pHSP20 (NH₂-βAYARRAAARQARAPRKS*LWALGRPLA-COOH (SEQ IDNO:308) (50 μM) and incubated for 24 hours. The cells were fixed andstained with hematoxylin. The number of cells migrating into a 1 cm²scratched area were counted as an index for migration. In additionalexperiments, the migration of A10 cells was determined in a Boydenchamber assay.

FIG. 9. A10 cells were serum starved for 3 days. The cells were thentreated with media containing 10% fetal bovine serum, PTD-pHSP20(NH₂-βAYARRAAARQARAWLRRAS*APLPGLK-COOH (SEQ ID NO:307), orPTD-scrambled-pHSP20 (NH₂-βAYARRAAARQARAPRKS*LWALGRPLA-COOH (SEQ IDNO:308) (50 μM). After 24 hours cell counts were performed.

DETAILED DESCRIPTION OF THE INVENTION

Within this application, unless otherwise stated, the techniquesutilized may be found in any of several well-known references such as:Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, SanDiego, Calif.), “Guide to Protein Purification” in Methods in Enzymology(M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.), Culture of Animal Cells: A Manual of BasicTechnique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.),and Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J.Murray, The Humana Press Inc., Clifton, N.J.)

In one aspect, the present invention provides polypeptides consisting ofan amino acid sequence according to general formula I:X1-X2-[X3-A(X4)APLP-X5-]_(u)-X6

wherein X1 is absent or is one or more molecules comprising one or morearomatic ring;

X2 is absent or comprises a transduction domain;

X3 is 0, 1, 2, 3, or 4 amino acids of the sequence WLRR (SEQ ID NO:1);

X4 is selected from the group consisting of S, T, Y, D, E,hydroxylysine, hydroxyproline, phosphoserine analogs and phosphotyrosineanalogs;

X5 is 0, 1, 2,or 3 amino acids of a sequence of genus Z1-Z2-Z3,

wherein Z1 is selected from the group consisting of G and D;

Z2 is selected from the group consisting of L and K; and

Z3 is selected from the group consisting of S and T;

X6 is absent or comprises a transduction domain; and

wherein u is 1-5.

Both single letter and three letter amino acid abbreviations are usedwithin the application. As used herein, “norL” means norleucine and“Orn” means ornithine.

The term “polypeptide” is used in its broadest sense to refer to asequence of subunit amino acids, amino acid analogs, or peptidomimetics.The subunits are linked by peptide bonds, except where noted (includingwhen the X2 position is a non-amino acid molecule that contains anaromatic ring). The polypeptides described herein may be chemicallysynthesized or recombinantly expressed.

Preferably, the polypeptides of the present invention are chemicallysynthesized. Synthetic polypeptides, prepared using the well knowntechniques of solid phase, liquid phase, or peptide condensationtechniques, or any combination thereof, can include natural andunnatural amino acids. Amino acids used for peptide synthesis may bestandard Boc (Nα-amino protected Nα-t-butyloxycarbonyl) amino acid resinwith the standard deprotecting, neutralization, coupling and washprotocols of the original solid phase procedure of Merrifield (1963, J.Am. Chem. Soc. 85:2149-2154), or the base-labile Nα-amino protected9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpinoand Han (1972, J. Org. Chem. 37:3403-3409). Both Fmoc and Boc Nα-aminoprotected amino acids can be obtained from Sigma, Cambridge ResearchBiochemical, or other chemical companies familiar to those skilled inthe art. In addition, the polypeptides can be synthesized with otherNα-protecting groups that are familiar to those skilled in this art.

Solid phase peptide synthesis may be accomplished by techniques familiarto those in the art and provided, for example, in Stewart and Young,1984, Solid Phase Synthesis, Second Edition, Pierce Chemical Co.,Rockford, Ill.; Fields and Noble, 1990, Int. J. Pept. Protein Res.35:161-214, or using automated synthesizers. The polypeptides of theinvention may comprise D-amino acids (which are resistant to L-aminoacid-specific proteases in vivo), a combination of D- and L-amino acids,and various “designer” amino acids (e.g., β-methyl amino acids,Cα-methyl amino acids, and Nα-methyl amino acids, etc.) to conveyspecial properties. Synthetic amino acids include ornithine for lysine,and norleucine for leucine or isoleucine.

In addition, the polypeptides can have peptidomimetic bonds, such asester bonds, to prepare peptides with novel properties. For example, apeptide may be generated that incorporates a reduced peptide bond, i.e.,R₁—CH₂—NH—R₂, where R₁ and R₂ are amino acid residues or sequences. Areduced peptide bond may be introduced as a dipeptide subunit. Such apolypeptide would be resistant to protease activity, and would possessan extended half-live in vivo.

According to various embodiments of the polypeptides of general formulaI, the region [X3-A(X4)APLP-X5-]_(u) may be present in 1, 2, 3, 4, or 5copies. In a preferred embodiment, it is present in 1 copy. In otherembodiments, it is present in multiple copies to provide increasedefficacy for use of the polypeptides for inhibiting one or more ofsmooth muscle cell proliferation, smooth muscle cell migration, andsmooth muscle spasm, and/or also for promoting smooth musclevasorelaxation.

According to various embodiments of the polypeptides of general formulaI, X4 is S, T, Y, D E, a phosphoserine mimic, or a phosphotyrosinemimic. It is more preferred that X4 is S, T, or Y; more preferred thatX4 is S or T, and most preferred that X4 is S. In these embodimentswhere X4 is S, T, or Y, it is most preferred that X4 is phosphorylated.When X4 is D or E, these residues have a negative charge that mimics thephosphorylated state. The polypeptides of the invention are optimallyeffective in the methods of the invention when X4 is phosphorylated, isa phosphoserine or phosphotyrosine mimic, or is another mimic of aphosphorylated amino acid residue, such as a D or E residue. Examples ofphosphoserine mimics include, but are not limited to, sulfoserine, aminoacid mimics containing a methylene substitution for the phosphateoxygen, 4-phosphono(difluoromethyl)phenylanaline, andL-2-amino-4-(phosphono)-4,4-difuorobutanoic acid. Other phosphoserinemimics can be made by those of skill in the art; for example, see Otakaet al., Tetrahedron Letters 36:927-930 (1995). Examples ofphosphotyrosine mimics include, but are not limited to,phosphonomethylphenylalanine, difluorophosphonomethylphenylalanine,fluoro-O-malonyltyrosine and O-malonyltyrosine. (See, for example,Akamatsu et. al., Bioorg Med Chem 1997 January;5(1):157-63).

In another preferred embodiment, X1 is one or more molecules comprisingan aromatic ring. In one preferred embodiment, the one or moleculescomprising an aromatic ring are amino acids, and X1 is (F/Y/W), wherein“z” is 1-5 amino acids. Thus, for example, X1 can be 1 or 2 amino acidresidues of any combination of F, Y, and W, such as F, FF, Y, YY, W, WW,FY, FW, YF, YW, WY, and WF. Alternatively, X1 an be a 3, 4, or 5 aminoacid combination of F, Y, and W. In another preferred embodiment, themolecule comprising an aromatic ring is selected from the group ofmolecules comprising one or more aromatic rings which can optionally besubstituted with halogen, lower alkyl, lower alkylthio, trifluoromethyl,lower acyloxy, aryl, and heteroaryl. In a most preferred embodiment, theone or more molecule comprising one or more aromatic ring comprise9-fluorenylmethyl (Fm). Examples of such molecules include, but are notlimited to 9-fluorenylmethylcarbonyl, 9-fluorenylmethylcarbamates,9-fluorenylmethylcarbonates, 9-fluorenylmethyl esters,9-fluorenylmethylphosphates, and S-9-fluorenylmethyl thioethers. Inembodiments wherein the molecule comprising an aromatic ring is not anamino acid, it can be attached to the polypeptide by methods known inthe art, including but not limited to, standard Fmoc protectionchemistry employed in peptide synthesis.

According to various embodiments of the polypeptides of general formulaI, X3 is 0, 1, 2, 3, or 4 amino acids of the sequence WLRR (SEQ IDNO:1). If X3 consists of only one amino acid of the sequence, an “R” ispresent, since it is the carboxy-terminal amino acid of the sequence andit would be present at the amino terminus of the rest of the A(X4)APLP(SEQ ID NO: 2) sequence. If X3 consists of two amino acids of WLRR (SEQID NO:1), then the two amino acids added will be “RR”. Other variationswill be apparent to one of skill in the art based on the teachingsherein.

Similarly, variations in the residues that can make up X5 will beapparent to one of skill in the art based on the teachings herein.

Thus, according to these various aspects, a representative sample ofpolypeptides according to general formula I include, but are not limitedto the following: (ASAPLP)_(u) (SEQ ID NO:3); (ATAPLP)_(u) (SEQ IDNO:4); (RASAPLP)_(u) (SEQ ID NO:5); (RATAPLP)_(u) (SEQ ID NO:6);(AYAPLP)_(u) (SEQ ID NO:7); (RAYAPLP)_(u) (SEQ ID NO:8);(RRASAPLP)_(u)(SEQ ID NO:9); (LRRASAPLP)_(u) (SEQ ID NO:10); (WLRRASAPLP)_(u); (SEQ IDNO:11) (RRATAPLP)_(u) (SEQ ID NO:12); (LRRATAPLP)_(u) (SEQ ID NO:13);(WLRRATAPLP)_(u)(SEQ ID NO:14); (RRAYAPLP)_(u) (SEQ ID NO:15);(LRRAYAPLP)_(u) (SEQ ID NO:16); (WLRRAYAPLP)_(u) (SEQ ID NO:17);(RRASAPLPG)_(u) (SEQ ID NO:18); (RRASAPLPD)_(u) (SEQ ID NO:19);(RRASAPLPGL)_(u) (SEQ ID NO:20); (RRASAPLPGK)_(u) (SEQ ID NO:21);(RRASAPLPDL)_(u) (SEQ ID NO:22); (RRASAPLPDK)_(u) (SEQ ID NO:23);(RRASAPLPGLS)_(u) (SEQ ID NO:24); (RRASAPLPGLT)_(u) (SEQ ID NO:25);(RRASAPLPGKS)_(u) (SEQ ID NO:26); (RRASAPLPGKT)_(u) (SEQ ID NO:27);(RRASAPLPDLS)_(u) (SEQ ID NO:28); RRASAPLPDLT)_(u) (SEQ ID NO:29);(RRASAPLPDKS)_(u) (SEQ ID NO:30); (RRASAPLPDKT)_(u) (SEQ ID NO:31);(LRRASAPLPG)_(u) (SEQ ID NO:32); (LRRASAPLPD)_(u) (SEQ ID NO:33);(LRRASAPLPGL)_(u) (SEQ ID NO:34); (LRRASAPLPGK)_(u) (SEQ ID NO:35);(LRRASAPLPDL)_(u) (SEQ ID NO:36); (LRRASAPLPDK)_(u) (SEQ ID NO:37);(LRRASAPLPGLS)_(u) (SEQ ID NO:38); (LRRASAPLPGLT)_(u) (SEQ ID NO:39);(LRRASAPLPGKS)_(u) (SEQ ID NO:40); (LRRASAPLPGKT)_(u) (SEQ ID NO:41);(LRRASAPLPDLS)_(u) (SEQ ID NO:42); (LRRASAPLPDLT)_(u) (SEQ ID NO:43);(LRRASAPLPDKS)_(u) (SEQ ID NO:44); (LRRASAPLPDKT)_(u) (SEQ ID NO:45);(WLRRASAPLPG)_(u) (SEQ ID NO:46); (WLRRASAPLPD)_(u) (SEQ ID NO:47);(WLRRASAPLPGL)_(u) (SEQ ID NO:48); (WLRRASAPLPGK)_(u) (SEQ ID NO:49);(WLRRASAPLPDL)_(u) (SEQ ID NO:50); (WLRRASAPLPDK)_(u) (SEQ ID NO:51);(WLRRASAPLPGLS)_(u) (SEQ ID NO:52); (WLRRASAPLPGLT)_(u) (SEQ ID NO:53);(WLRRASAPLPGKS)_(u) (SEQ ID NO:54); (WLRRASAPLPGKT)_(u) (SEQ ID NO:55);(WLRRASAPLPDLS)_(u) (SEQ ID NO:56); (WLRRASAPLPDLT)_(u) (SEQ ID NO:57);(WLRRASAPLPDKS)_(u) (SEQ ID NO:58); (WLRRASAPLPDKT)_(u) (SEQ ID NO:59);(RRATAPLPG)_(u) (SEQ ID NO:60); (RRATAPLPD)_(u) (SEQ ID NO:61);(RRATAPLPGL)_(u) (SEQ ID NO:62); (RRATAPLPGK)_(u) (SEQ ID NO:63);(RRATAPLPDL)_(u) (SEQ ID NO:64); (RRATAPLPDK)_(u) (SEQ ID NO:65);(RRATAPLPGLS)_(u) (SEQ ID NO:66); (RRATAPLPGLT)_(u) (SEQ ID NO:67);(RRATAPLPGKS)_(u) (SEQ ID NO:68); (RRATAPLPGKT)_(u) (SEQ ID NO:69);(RRATAPLPDLS)_(u) (SEQ ID NO:70); (RRATAPLPDLT)_(u) (SEQ ID NO:71);(RRATAPLPDKS)_(u) (SEQ ID NO:72); (RRATAPLPDKT)_(u) (SEQ ID NO:73);(LRRATAPLPG)_(u) (SEQ ID NO:74); (LRRATAPLPD)_(u) (SEQ ID NO:75);(LRRATAPLPGL)_(u) (SEQ ID NO:76); (LRRATAPLPGK)_(u) (SEQ ID NO:77);(LRRATAPLPDL)_(u) (SEQ ID NO:78); (LRRATAPLPDK)_(u) (SEQ ID NO:79);(LRRATAPLPGLS)_(u) (SEQ ID NO:80); (LRRATAPLPGLT)_(u) (SEQ ID NO:81);(LRRATAPLPGKS)_(u) (SEQ ID NO:82); (LRRATAPLPGKT)_(u) (SEQ ID NO:83);(LRRATAPLPDLS)_(u) (SEQ ID NO:84); (LRRATAPLPDLT)_(u) (SEQ ID NO:85);(LRRATAPLPDKS)_(u) (SEQ ID NO:86); (LRRATAPLPDKT)_(u) (SEQ ID NO:87);(WLRRATAPLPG)_(u) (SEQ ID NO:88); (WLRRATAPLPD)_(u) (SEQ ID NO:89);(WLRRATAPLPGL)_(u) (SEQ ID NO:90); (WLRRATAPLPGK)_(u) (SEQ ID NO:91);(WLRRATAPLPDL)_(u) (SEQ ID NO:92); (WLRRATAPLPDK)_(u) (SEQ ID NO:93);(WLRRATAPLPGLS)_(u) (SEQ ID NO:94); (WLRRATAPLPGLT)_(u) (SEQ ID NO:95);(WLRRATAPLPGKS)_(u) (SEQ ID NO:96); (WLRRATAPLPGKT)_(u) (SEQ ID NO:97);(WLRRATAPLPDLS)_(u) (SEQ ID NO:98); (WLRRATAPLPDLT)_(u) (SEQ ID NO:99);(WLRRATAPLPDKS)_(u) (SEQ ID NO:100); (WLRRATAPLPDKT)_(u) (SEQ IDNO:101); (RRAYAPLPG)_(u) (SEQ ID NO:102); (RRAYAPLPD)_(u) (SEQ IDNO:103); (RRAYAPLPGL)_(u) (SEQ ID NO:104); (RRAYAPLPGK)_(u) (SEQ IDNO:105); (RRAYAPLPDL)_(u) (SEQ ID NO:106); (RRAYAPLPDK)_(u) (SEQ IDNO:107); (RRAYAPLPGLS)_(u) (SEQ ID NO:108); (RRAYAPLPGLT)_(u) (SEQ IDNO:109); (RRAYAPLPGKS)_(u) (SEQ ID NO:110; (RRAYAPLPGKT)_(u) (SEQ IDNO:111); (RRAYAPLPDLS)_(u) (SEQ ID NO:112); (RRAYAPLPDLT)_(u) (SEQ IDNO:113); (RRAYAPLPDKS)_(u) (SEQ ID NO:114); (RRAYAPLPDKT)_(u) (SEQ IDNO:115); (LRRAYAPLPG)_(u) (SEQ ID NO:116); (LRRAYAPLPD)_(u) (SEQ IDNO:117); (LRRAYAPLPGL)_(u) (SEQ ID NO:118); (LRRAYAPLPGK)_(u) (SEQ IDNO:119); (LRRAYAPLPDL)_(u) (SEQ ID NO:120); (LRRAYAPLPDK)_(u) (SEQ IDNO:121); (LRRAYAPLPGLS)_(u) (SEQ ID NO:122); (LRRAYAPLPGLT)_(u) (SEQ IDNO:123); (LRRAYAPLPGKS)_(u) (SEQ ID NO:124); (LRRAYAPLPGKT)_(u) (SEQ IDNO:125); (LRRAYAPLPDLS)_(u) (SEQ ID NO:126); (LRRAYAPLPDLT)_(u) (SEQ IDNO:127); (LRRAYAPLPDKS)_(u) (SEQ ID NO:128); (LRRAYAPLPDKT)_(u) (SEQ IDNO:129); (WLRRAYAPLPG)_(u) (SEQ ID NO:130); (WLRRAYAPLPD)_(u) (SEQ IDNO:131); (WLRRAYAPLPGL)_(u) (SEQ ID NO:132); (WLRRAYAPLPGK)_(u) (SEQ IDNO:133); (WLRRAYAPLPDL)_(u) (SEQ ID NO:134); (WLRRAYAPLPDK)_(u) (SEQ IDNO:135); (WLRRAYAPLPGLS)_(u) (SEQ ID NO:136); (WLRRAYAPLPGLT)_(u) (SEQID NO:137); (WLRRAYAPLPGKS)_(u) (SEQ ID NO:138); (WLRRAYAPLPGKT)_(u)(SEQ ID NO:139); (WLRRAYAPLPDLS)_(u) (SEQ ID NO:140);(WLRRAYAPLPDLT)_(u) (SEQ ID NO:141); (WLRRAYAPLPDKS)_(u) (SEQ IDNO:142); and (WLRRAYAPLPDKT)_(u) (SEQ ID NO:143); ((F/Y/W)RRASAPLP)_(u)(SEQ ID NO:144); ((F/Y/W)LRRASAPLP)_(u) (SEQ ID NO:145);((F/Y/W)WLRRASAPLP)_(u) ; (SEQ ID NO:146) ((F/Y/W)RRATAPLP)_(u) (SEQ IDNO:147); ((F/Y/W)LRRATAPLP)_(u) (SEQ ID NO:148); ((F/Y/W)WLRRATAPLP)_(u)(SEQ ID NO:149); ((FIY/W)RRAYAPLP)_(u) (SEQ ID NO:150);((F/Y/W)LRRAYAPLP)_(u) (SEQ ID NO:151); ((F/Y/W)WLRRAYAPLP)_(u) (SEQ IDNO:152); ((F/Y/W)RRASAPLPG)_(u) (SEQ ID NO:153); ((F/Y/W)RRASAPLPD)_(u)(SEQ ID NO:154); ((F/Y/W)RRASAPLPGL)_(u) (SEQ ID NO:155);((F/Y/W)RRASAPLPGK)_(u) (SEQ ID NO:156); ((F/Y/W)RRASAPLPDL)_(u) (SEQ IDNO:157); ((F/Y/W)RRASAPLPDK)_(u) (SEQ ID NO:158);((F/Y/W)RRASAPLPGLS)_(u) (SEQ ID NO:159); ((F/Y/W)RRASAPLPGLT)_(u) (SEQID NO:160); ((F/Y/W)RRASAPLPGKS)_(u) ; (SEQ ID NO:161);((F/Y/W)RRASAPLPGKT)_(u) (SEQ ID NO:162); ((F/Y/W)RRASAPLPDLS)_(u) (SEQID NO:163); ((F/Y/W)RRASAPLPDLT)_(u) (SEQ ID NO:164);((F/Y/W)RRASAPLPDKS)_(u) (SEQ ID NO:165); ((F/Y/W)RRASAPLPDKT)_(u) (SEQID NO:166); ((F/Y/W)LRRASAPLPG)_(u) (SEQ ID NO:167);((F/Y/W)LRRASAPLPD)_(u) (SEQ ID NO:168); ((F/Y/W))LRRASAPLPGL)_(u) (SEQID NO:169); ((F/Y/W)LRRASAPLPGK)_(u) (SEQ ID NO:170);((F/Y/W)LRRASAPLPDL)_(u) (SEQ ID NO:171); ((F/Y/W)LRRASAPLPDK)_(u) (SEQID NO:172); ((F/Y/W)LRRASAPLPGLS)_(u) (SEQ ID NO:173);((F/Y/W)LRRASAPLPGLT)_(u) (SEQ ID NO:174); ((F/Y/W)LRRASAPLPGKS)_(u)(SEQ ID NO:175); ((F/Y/W)LRRASAPLPGKT)_(u) (SEQ ID NO:176);((F/Y/W)LRRASAPLPDLS)_(u) (SEQ ID NO:177); ((F/Y/W)LRRASAPLPDLT)_(u)(SEQ ID NO:178); ((F/Y/W)LRRASAPLPDKS)_(u) (SEQ ID NO:179);((F/Y/W)LRRASAPLPDKT)_(u) (SEQ ID NO:180); ((F/Y/W)WLRRASAPLPG)_(u) (SEQID NO:181); ((F/Y/W)WLRRASAPLPD)_(u) (SEQ ID NO:182);((F/Y/W)WLRRASAPLPGL)_(u) (SEQ ID NO:183); ((F/Y/W)WLRRASAPLPGK)_(u)(SEQ ID NO:184); ((F/Y/W)WLRRASAPLPDL)_(u) (SEQ ID NO:185);((F/Y/W)WLRRASAPLPDK)_(u) (SEQ ID NO:186); ((F/Y/W)WLRRASAPLPGLS)_(u)(SEQ ID NO:187); ((F/Y/W)WLRRASAPLPGLT)_(u) (SEQ ID NO:188);((F/Y/W)WLRRASAPLPGKS)_(u) (SEQ ID NO:189); ((F/Y/W)WLRRASAPLPGKT)_(u)(SEQ ID NO:190); ((F/Y/W)WLRRASAPLPDLS)_(u) (SEQ ID NO:191);((F/Y/W)WLRRASAPLPDLT)_(u) (SEQ ID NO:192); ((F/Y/W)WLRRASAPLPDKS)_(u)(SEQ ID NO:193); ((F/Y/W)WLRRASAPLPDKT)_(u) (SEQ ID NO:194);((F/Y/W)RRATAPLPG)_(u) (SEQ ID NO:195); ((F/Y/W)RRATAPLPD)_(u) (SEQ IDNO:196); ((F/Y/W)RRATAPLPGL)_(u) (SEQ ID NO:197);((F/Y/W)RRATAPLPGK)_(u) (SEQ ID NO:198); ((F/Y/W)RRATAPLPDL)_(u) (SEQ IDNO:199); ((F/Y/W)RRATAPLPDK)_(u) (SEQ ID NO:200);((F/Y/W)RRATAPLPGLS)_(u) (SEQ ID NO:201); ((F/Y/W)RRATAPLPGLT)_(u) (SEQID NO:202); ((F/Y/W)RRATAPLPGKS)_(u) (SEQ ID NO:203);((F/Y/W)RRATAPLPGKT)_(u) (SEQ ID NO:204); ((F/Y/W)RRATAPLPDLS)_(u) (SEQID NO:205); ((F/Y/W)RRATAPLPDLT)_(u) (SEQ ID NO:206);((F/Y/W)RRATAPLPDKS)_(u) (SEQ ID NO:207); ((F/Y/W)RRATAPLPDKT)_(u) (SEQID NO:208); ((F/Y/W)LRRATAPLPG)_(u) (SEQ ID NO:209);((F/Y/W)LRRATAPLPD)_(u) (SEQ ID NO:210); ((F/Y/W)LRRATAPLPGL)_(u) (SEQID NO:211); ((F/Y/W)LRRATAPLPGK)_(u) (SEQ ID NO:212);((F/Y/W)LRRATAPLPDL)_(u) (SEQ ID NO:213); ((F/Y/W)LRRATAPLPDK)_(u) (SEQID NO:214); ((F/Y/W)LRRATAPLPGLS)_(u) (SEQ ID NO:215);((F/Y/W)LRRATAPLPGLT)_(u) (SEQ ID NO:216); ((F/Y/W)LRRATAPLPGKS)_(u)(SEQ ID NO:217); ((F/Y/W)LRRATAPLPGKT)_(u) (SEQ ID NO:218);((F/Y/W)LRRATAPLPDLS)_(u) (SEQ ID NO:219); ((F/Y/W)LRRATAPLPDLT)_(u)(SEQ ID NO:220); ((F/Y/W)LRRATAPLPDKS)_(u) (SEQ ID NO:221);((F/Y/W)LRRATAPLPDKT)_(u) (SEQ ID NO:222); ((FIYIW)WLRRATAPLPG)_(u) (SEQID NO:223); ((F/Y/W)WLRRATAPLPD)_(u) (SEQ ID NO:224);((F/Y/W)WLRRATAPLPGL)_(u) (SEQ ID NO:225); ((F/Y/W)WLRRATAPLPGK)_(u)(SEQ ID NO:226); ((F/Y/W)WLRRATAPLPDL)_(u) (SEQ ID NO:227);((F/Y/W)WLRRATAPLPDK)_(u) (SEQ ID NO:228); ((F/Y/W)WLRRATAPLPGLS)_(u)(SEQ ID NO:229); ((F/Y/W)WLRRATAPLPGLT)_(u) (SEQ ID NO:230);((F/Y/W)WLRRATAPLPGKS)_(u) (SEQ ID NO:231); ((F/Y/W)WLRRATAPLPGKT)_(u)(SEQ ID NO:232); ((F/Y/W)WLRRATAPLPDLS)_(u) (SEQ ID NO:233);((F/Y/W)WLRRATAPLPDLT)_(u) (SEQ ID NO:234); ((F/Y/W)WLRRATAPLPDKS)_(u)(SEQ ID NO:235); ((F/Y/W)WLRRATAPLPDKT)_(u) (SEQ ID NO:236);((F/Y/W)RRAYAPLPG)_(u) (SEQ ID NO:237); ((F/Y/W)RRAYAPLPD)_(u) (SEQ IDNO:238); ((F/Y/W)RRAYAPLPGL)_(u) (SEQ ID NO:239);((F/Y/W)RRAYAPLPGK)_(u) (SEQ ID NO:240); ((F/Y/W)RRAYAPLPDL)_(u) (SEQ IDNO:241); ((F/Y/W)RRAYAPLPDK)_(u) (SEQ ID NO:242);((F/Y/W)RRAYAPLPGLS)_(u) (SEQ ID NO:243); ((F/Y/W)RRAYAPLPGLT)_(u) (SEQID NO:244); ((F/Y/W)RRAYAPLPGKS)_(u) (SEQ ID NO:245);((F/Y/W)RRAYAPLPGKT)_(u) (SEQ ID NO:246); ((F/Y/W)RRAYAPLPDLS)_(u) (SEQID NO:247); ((F/Y/W)RRAYAPLPDLT)_(u) (SEQ ID NO:248);((F/Y/W)RRAYAPLPDKS)_(u) (SEQ ID NO:249); ((F/Y/W)RRAYAPLPDKT)_(u) (SEQID NO:250); ((F/Y/W)LRRAYAPLPG)_(u) (SEQ ID NO:251);((F/Y/W)LRRAYAPLPD)_(u) (SEQ ID NO:252); ((F/Y/W)LRRAYAPLPGL)_(u) (SEQID NO:253); ((F/Y/W)LRRAYAPLPGK)_(u) (SEQ ID NO:254);((F/Y/W)LRRAYAPLPDL)_(u) (SEQ ID NO:255); ((F/Y/W)LRRAYAPLPDK)_(u) (SEQID NO:256); ((F/Y/W)LRRAYAPLPGLS)_(u) (SEQ ID NO:257);((F/Y/W)LRRAYAPLPGLT)_(u) (SEQ ID NO:258); ((F/Y/W)LRRAYAPLPGKS)_(u)(SEQ ID NO:259); ((F/Y/W)LRRAYAPLPGKT)_(u) (SEQ ID NO:260);((F/Y/W)LRRAYAPLPDLS)_(u) (SEQ ID NO:261); ((F/Y/W)LRRAYAPLPDLT)_(u)(SEQ ID NO:262); ((F/Y/W)LRRAYAPLPDKS)_(u) (SEQ ID NO:263);((F/Y/W)LRRAYAPLPDKT)_(u) (SEQ ID NO:264); ((F/Y/W)WLRRAYAPLPG)_(u) (SEQID NO:265); ((F/Y/W)WLRRAYAPLPD)_(u) (SEQ ID NO:266);((F/Y/W)WLRRAYAPLPGL)_(u) (SEQ ID NO:267); ((F/Y/W)WLRRAYAPLPGK)_(u)(SEQ ID NO:268); ((F/Y/W)WLRRAYAPLPDL)_(u) (SEQ ID NO:269);((F/Y/W)WLRRAYAPLPDK)_(u) (SEQ ID NO:270); ((F/Y/W)WLRRAYAPLPGLS)_(u)(SEQ ID NO:271); ((F/Y/W)WLRRAYAPLPGLT)_(u) (SEQ ID NO:272);((F/Y/W)WLRRAYAPLPGKS)_(u) (SEQ ID NO:273); ((F/Y/W)WLRRAYAPLPGKT)_(u)(SEQ ID NO:274); ((F/Y/W)WLRRAYAPLPDLS)_(u) (SEQ ID NO:275);((F/Y/W)WLRRAYAPLPDLT)_(u) (SEQ ID NO:276); ((F/Y/W)WLRRAYAPLPDKS)_(u)(SEQ ID NO:277); and ((F/Y/W)WLRRAYAPLPDKT)_(u) (SEQ ID NO:278) wherein“u” is as defined above, and (F/Y/W) means that the residue is selectedfrom F, Y, and W. Other specific polypeptides falling within the scopeof general formula I will be readily apparent to one of skill in the artbased on the teachings herein.

In a further embodiment, the polypeptides of the present inventionconsist of a combination of different sequences from the region[X3-A(X4)APLP-X5-]_(u) . In this embodiment, for example, thepolypeptide can consist of 1 copy of SEQ ID NO:9 and 1 copy of SEQ IDNO:143. In a different example, the polypeptide could consist of 2copies of SEQID NO:200 and 3 copies of SEQ ID NO:62. It will be apparentto one of skill in the art that many such combinations are possiblebased on the teachings of the present invention.

In a preferred embodiment, at least one of X2 and X6 comprises atransduction domain. As used herein, the term “transduction domain”means one or more amino acid sequence or any other molecule that cancarry the active domain across cell membranes. These domains can belinked to other polypeptides to direct movement of the linkedpolypeptide across cell membranes. In some cases the transducingmolecules do not need to be covalently linked to the active polypeptide(for example, see sequence ID 291). In a preferred embodiment, thetransduction domain is linked to the rest of the polypeptide via peptidebonding. (See, for example, Cell 55: 1179-1188, 1988; Cell 55:1189-1193, 1988; Proc Natl Acad Sci USA 91: 664-668, 1994; Science 285:1569-1572, 1999; J Biol Chem 276: 3254-3261, 2001; and Cancer Res 61:474-477, 2001) In this embodiment, any of the polypeptides as describedabove would include at least one transduction domain. In a furtherembodiment, both X2 and X6 comprise transduction domains. In a furtherpreferred embodiment, the transduction domain(s) is/are selected fromthe group consisting of (R)₄₋₉ (SEQ ID NO:279); GRKKRRQRRRPPQ (SEQ IDNO:280); AYARAAARQARA (SEQ ID NO:281);DAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:282);GWTLNSAGYLLGLINLKALAALAKKIL (SEQ ID NO:283); PLSSIFSRIGDP (SEQ IDNO:284); AAVALLPAVLLALLAP (SEQ ID NO:285); AAVLLPVLLAAP (SEQ ID NO:286);VTVLALGALAGVGVG (SEQ ID NO:287); GALFLGWLGAAGSTMGAWSQP (SEQ ID NO:288);GWTLNSAGYLLGLINLKALAALAKKIL (SEQ ID NO:289); KLALKLALKALKAALKLA (SEQ IDNO:290); KETWWETWWTEWSQPKKKRKV (SEQ ID NO:291); KAFAKLAARLYRKAGC (SEQ IDNO:292); KAFAKLAARLYRAAGC (SEQ ID NO:293); AAFAKLAARLYRKAGC (SEQ IDNO:294); KAFAALAARLYRKAGC (SEQ ID NO:295); KAFAKLAAQLYRKAGC (SEQ IDNO:296), and AGGGGYGRKKRRQRRR (SEQ ID NO:306).

In another embodiment, the present invention provides a polypeptidecomprising a sequence according to general formula II:X1-X2-[X3-A(X4)APLP-X5]_(u) -X6wherein X1 is absent or is one or more molecules comprising one or morearomatic ring;

X2 is absent or comprises a cell transduction domain;

X3 is 0-14 amino acids of the sequence of heat shock protein 20 betweenresidues 1 and 14 of SEQ ID NO:297;

X4 is selected from the group consisting of S, T, Y, D, E,hydroxylysine, hydroxyproline, phosphoserine analogs and phosphotyrosineanalogs;

X5 is 0-140 amino acids of heat shock protein 20 between residues 21 and160 of SEQ ID NO:297;

X6 is absent or comprises a cell transduction domain; and

wherein at least one of X2 and X6 comprise a transduction domain.

Thus, in various preferred embodiments of the polypeptide of generalformula II, X4 is S, T, Y, D, E, a phosphoserine analog, or aphosphotyrosine analog. In a preferred embodiment, X4 is S, T, or Y. Ina more preferred embodiment, X4 is S or T. In a most preferredembodiment, X4 is S.

In these embodiments where X4 is S, T, or Y, it is most preferred thatX4 is phosphorylated. When X4 is D or E, these residues have a negativecharge that mimics the phosphorylated state. The polypeptides of theinvention are optimally effective in the methods of the invention whenX4 is phosphorylated, is a phosphoserine or phosphotyrosine mimic, or isanother mimic of a phosphorylated amino acid residue, such as a D or Eresidue.

In a further preferred embodiment, X1 is one or more moleculescomprising one or more aromatic ring, as disclosed above, with preferredembodiments as disclosed above.

According to these embodiments, the polypeptide comprises at least onetransduction domain. In a further embodiment, both X2 and X6 comprise atransduction domain. Preferred embodiments of such transduction domainsare as described above.

One preferred embodiment of the polypeptide of general formula IIcomprises full length HSP20 (X1-X2-SEQ ID NO:297-X6).

Met Glu Ile Pro Val Pro Val Gln Pro Ser Trp Leu Arg Arg Ala Ser Ala ProLeu Pro Gly Leu Ser Ala Pro Gly Arg Leu Phe Asp Gln Arg Phe Gly Glu GlyLeu Leu Glu Ala Glu Leu Ala Ala Leu Cys Pro Thr Thr Leu Ala Pro Tyr TyrLeu Arg Ala Pro Ser Val Ala Leu Pro Val Ala Gln Val Pro Thr Asp Pro GlyHis Phe Ser Val Leu Leu Asp Val Lys His Phe Ser Pro Glu Glu Ile Ala ValLys Val Val Gly Glu His Val Glu Val His Ala Arg His Glu Glu Arg Pro AspGlu His Gly Phe Val Ala Arg Glu Phe His Arg Arg Tyr Arg Leu Pro Pro GlyVal Asp Pro Ala Ala Val Thr Ser Ala Leu Ser Pro Glu Gly Val Leu Ser IleGln Ala Ala Pro Ala Ser Ala Gln Ala Pro Pro Pro Ala Ala Ala Lys. (SEQ IDNO:297)

Another preferred embodiment of the polypeptide of general formula IIcomprises full length HSP20 with the serine at position 16 substitutewith aspartic acid (X1-X2-SEQ ID NO:298-X6):

Met Glu Ile Pro Val Pro Val Gln Pro Ser Trp Leu Arg Arg Ala Asp Ala ProLeu Pro Gly Leu Ser Ala Pro Gly Arg Leu Phe Asp Gln Arg Phe Gly Glu GlyLeu Leu Glu Ala Glu Leu Ala Ala Leu Cys Pro Thr Thr Leu Ala Pro Tyr TyrLeu Arg Ala Pro Ser Val Ala Leu Pro Val Ala Gln Val Pro Thr Asp Pro GlyHis Phe Ser Val Leu Leu Asp Val Lys His Phe Ser Pro Glu Glu Ile Ala ValLys Val Val Gly Glu His Val Glu Val His Ala Arg His Glu Glu Arg Pro AspGlu His Gly Phe Val Ala Arg Glu Phe His Arg Arg Tyr Arg Leu Pro Pro GlyVal Asp Pro Ala Ala Val Thr Ser Ala Leu Ser Pro Glu Gly Val Leu Ser IleGln Ala Ala Pro Ala Ser Ala Gln Ala Pro Pro Pro Ala Ala Ala Lys. (SEQ IDNO:298)

Another preferred embodiment of the polypeptide of general formula IIcomprises full length HSP20 with the serine at position 16 substitutewith glutamic acid (X1-X2-SEQ ID NO:299-X6):

Met Glu Ile Pro Val Pro Val Gln Pro Ser Trp Leu Arg Arg Ala Glu Ala ProLeu Pro Gly Leu Ser Ala Pro Gly Arg Leu Phe Asp Gln Arg Phe Gly Glu GlyLeu Leu Glu Ala Glu Leu Ala Ala Leu Cys Pro Thr Thr Leu Ala Pro Tyr TyrLeu Arg Ala Pro Ser Val Ala Leu Pro Val Ala Gln Val Pro Thr Asp Pro GlyHis Phe Ser Val Leu Leu Asp Val Lys His Phe Ser Pro Glu Glu Ile Ala ValLys Val Val Gly Glu His Val Glu Val His Ala Arg His Glu Glu Arg Pro AspGlu His Gly Phe Val Ala Arg Glu Phe His Arg Arg Tyr Arg Leu Pro Pro GlyVal Asp Pro Ala Ala Val Thr Ser Ala Leu Ser Pro Glu Gly Val Leu Ser IleGln Ala Ala Pro Ala Ser Ala Gln Ala Pro Pro Pro Ala Ala Ala Lys. (SEQ IDNO:299)

Other preferred embodiments according to general formula II are thepeptides disclosed above as embodiments of general formula I with therequired transduction domain at either X2 or X6, or both. Still furtherpreferred embodiments according to general formula II are the following:

X1-X2-SEQ ID NO:300-X6, wherein (SEQ ID NO: 300) is Trp Leu Arg Arg AlaSer Ala Pro Leu Pro Gly Leu Lys;

X1-X2-SEQ ID NO:301-X6, wherein (SEQ ID NO: 301) is Trp Leu Arg Arg AlaAsp Ala Pro Leu Pro Gly Leu Lys;. and

X1-X2-SEQ ID NO:302-X6, wherein (SEQ ID NO: 302) is Trp Leu Arg Arg AlaGlu Ala Pro Leu Pro Gly Leu Lys.

In these embodiments of the polypeptides according to general formulaII, it is preferred that the polypeptides are phosphorylated, mostpreferably at residue 16, or contain phosphorylation mimics at theposition of amino acid residue 16.

In a further aspect, the present invention provides a composition,comprising one or more polypeptides of the present invention, and aninhibitor of HSP27. HSP27 is closely related to HSP20; the two proteinsoften co-exist in macromolecular aggregates, and both areactin-associated proteins. Increases in the phosphorylation of HSP27 areassociated with smooth muscle contraction, and transfection of cellswith dominant active phosphorylated mutants of HSP27 leads to stressfiber formation (Mol Cell Biol 15: 505-516, 1995). Furthermore,increases in the phosphorylation of HSP27 are associated with smoothmuscle cell migration. HSP20, in contrast, promotes vasorelaxation, andthe data presented herein demonstrates that phosphorylated analogues ofHSP20 lead to a loss of stress fiber formation, and inhibit smoothmuscle cell proliferation and migration (See the examples below). Thus,the data indicate that HSP20 and HSP27 have opposing functions.Therefore, the combined use of one or more polypeptides of the inventionand an inhibitor of HSP27 will have enhanced efficacy in carrying outthe methods of the invention for inhibiting smooth muscle cellproliferation and/or migration, for promoting smooth muscle relaxation,and for inhibiting smooth muscle spasm (see below).

As used herein, an “inhibitor” of HSP27 includes HSP27 antibodies,anti-sense HSP27 nucleic acids, or small molecule inhibitors of thephosphorylation of HSP27, such as SB203580 (available from SmithKlineBeecham).

The polypeptides may be subjected to conventional pharmaceuticaloperations such as sterilization and/or may contain conventionaladjuvants, such as preservatives, stabilizers, wetting agents,emulsifiers, buffers etc.

In another aspect, the present invention provides pharmaceuticalcompositions, comprising one or more of the polypeptides disclosedherein, and a pharmaceutically acceptable carrier. Such pharmaceuticalcompositions are especially useful for carrying out the methods of theinvention described below.

For administration, the polypeptides are ordinarily combined with one ormore adjuvants appropriate for the indicated route of administration.The compounds may be admixed with lactose, sucrose, starch powder,cellulose esters of alkanoic acids, stearic acid, talc, magnesiumstearate, magnesium oxide, sodium and calcium salts of phosphoric andsulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine,dextran sulfate, heparin-containing gels, and/or polyvinyl alcohol, andtableted or encapsulated for conventional administration. Alternatively,the compounds of this invention may be dissolved in saline, water,polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidalsolutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil,tragacanth gum, and/or various buffers. Other adjuvants and modes ofadministration are well known in the pharmaceutical art. The carrier ordiluent may include time delay material, such as glyceryl monostearateor glyceryl distearate alone or with a wax, or other materials wellknown in the art.

The polypeptides or pharmaceutical compositions thereof may beadministered by any suitable route, including orally, parentally, byinhalation spray, rectally, or topically in dosage unit formulationscontaining conventional pharmaceutically acceptable carriers, adjuvants,and vehicles. The term parenteral as used herein includes, subcutaneous,intravenous, intra-arterial, intramuscular, intrasternal,intratendinous, intraspinal, intracranial, intrathoracic, infusiontechniques or intraperitoneally. Preferred embodiments foradministration vary with respect to the condition being treated, and aredescribed in detail below.

The polypeptides may be made up in a solid form (including granules,powders or suppositories) or in a liquid form (e.g., solutions,suspensions, or emulsions). The polypeptides of the invention may beapplied in a variety of solutions. Suitable solutions for use inaccordance with the invention are sterile, dissolve sufficient amountsof the polypeptides, and are not harmful for the proposed application.

In another aspect, the present invention provides an isolated nucleicacid sequence encoding a polypeptide of the present invention.Appropriate nucleic acid sequences according to this aspect of theinvention will be apparent to one of skill in the art based on thedisclosure provided herein and the general level of skill in the art.One example of such a nucleic acid sequence is provided as SEQ IDNO:320.

In another aspect, the present invention provides an expression vectorcomprising DNA control sequences operably linked to the isolated nucleicacid molecules of the present invention, as disclosed above. “Controlsequences” operably linked to the nucleic acid sequences of theinvention are nucleic acid sequences capable of effecting the expressionof the nucleic acid molecules. The control sequences need not becontiguous with the nucleic acid sequences, so long as they function todirect the expression thereof. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the nucleic acid sequences and the promoter sequence canstill be considered “operably linked” to the coding sequence. Other suchcontrol sequences include, but are not limited to, polyadenylationsignals, termination signals, and ribosome binding sites.

Such expression vectors can be of any type known in the art, includingbut not limited plasmid and viral-based expression vectors.

In a further aspect, the present invention provides geneticallyengineered host cells comprising the expression vectors of theinvention. Such host cells can be prokaryotic cells or eukaryotic cells,and can be either transiently or stably transfected, or can betransduced with viral vectors.

In another aspect, the invention provides improved biomedical devices,wherein the biomedical devices comprise one or more of the polypeptidesof the present invention disposed on or in the biomedical device. In apreferred embodiment, the one or more polypeptides are phosphorylated,as discussed above.

As used herein, a “biomedical device” refers to a device to be implantedinto a subject, for example, a human being, in order to bring about adesired result. Particularly preferred biomedical devices according tothis aspect of the invention include, but are not limited to, stents,grafts, shunts, stent grafts, fistulas, angioplasty devices, ballooncatheters and any implantable drug delivery device.

As used herein, the term “grafts” refers to both natural and prostheticgrafts and implants. In a most preferred embodiment, the graft is avascular graft.

As used herein, the term “stent” includes the stent itself, as well asany sleeve or other component that may be used to facilitate stentplacement.

As used herein, “disposed on or in” means that the one or morepolypeptides can be either directly or indirectly in contact with anouter surface, an inner surface, or embedded within the biomedicaldevice. “Direct” contact refers to disposition of the polypeptidesdirectly on or in the device, including but not limited to soaking abiomedical device in a solution containing the one or more polypeptides,spin coating or spraying a solution containing the one or morepolypeptides onto the device, implanting any device that would deliverthe polypeptide, and administering the polypeptide through a catheterdirectly on to the surface or into any organ.

“Indirect” contact means that the one or more polypeptides do notdirectly contact the biomedical device. For example, the one or morepolypeptides may be disposed in a matrix, such as a gel matrix or aviscous fluid, which is disposed on the biomedical device. Such matricescan be prepared to, for example, modify the binding and releaseproperties of the one or more polypeptides as required.

In a further embodiment, the biomedical device further comprises aninhibitor of the small heat shock protein HSP27 disposed on or in thebiomedical device. In a preferred embodiment, such inhibitors areselected from HSP27 antibodies, anti-sense HSP27 nucleic acids, or smallmolecule inhibitors of the phosphorylation of HSP27, such as SB203580.

In another aspect, the invention provides methods for inhibiting smoothmuscle cell proliferation and/or migration, comprising contacting thesmooth muscle cells with an amount effective to inhibit smooth musclecell proliferation and/or migration of HSP20, or functional equivalentsthereof, such as one or more polypeptide according to general formula Ior II. In a most preferred embodiment, the one or more polypeptides arephosphorylated as disclosed above. In a further embodiment, the methodfurther comprises contacting the smooth muscle cells with an amounteffective to inhibit smooth muscle cell proliferation and/or migrationof an inhibitor of the small heat shock protein HSP27. In a furtherembodiment, the method further comprises contacting the cells with anamount of PKG sufficient to stimulate HSP20 phosphorylation, wherein thecontacting comprises transfecting the cells with an expression vectorthat is capable of directing the expression of PKG, or by transducingthe cells with a PKG-transduction domain chimera.

Intimal hyperplasia is a complex process that leads to graft failure,and is the most common cause of failure of arterial bypass grafts. Whileincompletely understood, intimal hyperplasia is mediated by a sequenceof events that include endothelial cell injury and subsequent vascularsmooth muscle proliferation and migration from the media to the intima.This process is associated with a phenotypic modulation of the smoothmuscle cells from a contractile to a synthetic phenotype. The“synthetic” smooth muscle cells secrete extracellular matrix proteins,which leads to pathologic narrowing of the vessel lumen leading to graftstenoses and ultimately graft failure. Such endothelial cell injury andsubsequent smooth muscle cell proliferation and migration into theintima also characterizes restenosis, most commonly after angioplasty toclear an obstructed blood vessel. As discussed below, HSP20, andfunctional equivalents thereof, such as the polypeptides of generalformula I and II, inhibit smooth muscle cell proliferation andmigration.

In this aspect, the method can be in vitro or in vivo. In oneembodiment, the method is in vitro, wherein a vein or arterial graft iscontacted with HSP20 or a functional equivalent(s) thereof, prior tografting in a patient, in order to inhibit smooth muscle cellproliferation and/or migration, and thus to inhibit subsequent intimalhyperplasia and stenosis after placement of the graft, which could leadto graft failure. This can be accomplished, for example, by deliveringthe recombinant expression vectors (most preferably a viral vector, suchas an adenoviral vector) of the invention to the site, and transfectingthe smooth muscle cells. More preferably, delivery into smooth musclecells is accomplished by using the polypeptides of general formula I orII that include at least one transduction domain to facilitate entryinto the smooth muscle cells. The examples below demonstrate the abilityof the polypeptides of the invention that contain at least onetransduction domain to be delivered into smooth muscle cells.

In a more preferred in vitro embodiment, the method comprises contactingthe graft with one or more of the polypeptides of the invention thatinclude at least one transduction domain. Upon placement of the graft,it is preferred that the subject receiving be treated systemically withheparin, as heparin has been shown to bind to protein transductiondomains and prevent them from transducing into cells. This approach willlead to localized protein transduction of the graft alone, and not intoperipheral tissues.

In various other preferred embodiments of this aspect, the method isperformed in vivo, and is used to treat or prevent a disorder selectedfrom the group consisting of intimal hyperplasia, stenosis, restenosis,and atherosclerosis. In these embodiments, the contacting may be direct,by contacting a blood vessel in a subject being treated with HSP20 or afunctional equivalent(s) thereof, such as the polypeptides according togeneral formula I or II. For example, a liquid preparation of HSP20 or afunctional equivalent(s) thereof, such as the polypeptides according togeneral formula I or II, can be forced through a porous catheter, orotherwise injected through a catheter to the injured site, or a gel orviscous liquid containing the one or more polypeptides could be spreadon the injured site. In these embodiment of direct delivery, it is mostpreferred that the HSP20 or a functional equivalent(s) thereof, such asthe polypeptides according to general formula I or II be delivered intosmooth muscle cells at the site of injury or intervention. This can beaccomplished, for example, by delivering the recombinant expressionvectors (most preferably a viral vector, such as an adenoviral vector)of the invention to the site. More preferably, delivery into smoothmuscle cells is accomplished by using the polypeptides of generalformula I or II that include at least one transduction domain tofacilitate entry into the smooth muscle cells. The examples belowdemonstrate the ability of the polypeptides of the invention thatcontain at least one transduction domain to be delivered into smoothmuscle cells.

In various other preferred embodiments of this aspect of the invention,the method is performed on a subject who has undergone, is undergoing,or will undergo a procedure selected from the group consisting ofangioplasty, vascular stent placement, endarterectomy, atherectomy,bypass surgery (such as coronary artery bypass surgery; peripheralvascular bypass surgeries), vascular grafting, organ transplant,prosthetic device implanting, microvascular reconstructions, plasticsurgical flap construction, and catheter emplacement.

In a further embodiment of this aspect of the invention, the method isused to treat smooth muscle cell tumors. In a preferred embodiment, thetumor is a leiomyosarcoma, which is defined as a malignant neoplasm thatarises from muscle. Since leiomyosarcomas can arise from the walls ofboth small and large blood vessels, they can occur anywhere in the body,but peritoneal, uterine, and gastro-intestinal (particularly esophageal)leiomyosarcomas are more common. Alternatively, the smooth muscle tumorcan be a leiomyoma, a non-malignant smooth muscle neoplasm. In a mostpreferred embodiment, the one or more polypeptides are phosphorylated asdisclosed above. In a further embodiment, the method further comprisescontacting the smooth muscle cells with an amount effective to inhibitsmooth muscle cell proliferation and/or migration of an inhibitor of thesmall heat shock protein HSP27.

As further discussed in the examples below, HSP20, and functionalequivalents thereof, such as the polypeptides of general formula I andII, also disrupt actin stress fiber formation and adhesion plaques, eachof which have been implicated in intimal hyperplasia. The data furtherdemonstrate a direct inhibitory effect of the polypeptides of thepresent invention on intimal hyperplasia. Thus, in another aspect, thepresent invention further provides methods for treating or inhibitingone or more disorder selected from the group consisting of intimalhyperplasia, stenosis, restenosis, and atherosclerosis, comprisingcontacting a subject in need thereof with an amount effective to treator inhibit intimal hyperplasia, stenosis, restenosis, and/oratherosclerosis of HSP20, or a functional equivalent thereof, such asone or more polypeptides according to general formula I or II. Deliveryof the HSP20, or a functional equivalent thereof, such as one or morepolypeptides according to general formula I or II, in this aspect are asdisclosed above. In a most preferred embodiment, the one or morepolypeptides are phosphorylated as disclosed above. In a furtherembodiment, the method further comprises contacting the smooth musclecells with an amount effective to inhibit smooth muscle cellproliferation and/or migration of an inhibitor of the small heat shockprotein HSP27.

In various other preferred embodiments of this aspect of the invention,the method is performed on a subject who has undergone, is undergoing,or will undergo a procedure selected from the group consisting ofangioplasty, vascular stent placement, endarterectomy, atherectomy,bypass surgery, vascular grafting, microvascular reconstructions,plastic surgical flap construction, organ transplant, and catheteremplacement.

In a further aspect, the present invention provides methods to treatsmooth muscle cell tumors, comprising administering to a subject in needthereof of an amount effective of HSP20, or a functional equivalentthereof, such as one or more polypeptides according to general formula Ior II, to inhibit smooth muscle tumor growth and/or metastasis. In apreferred embodiment, the tumor is a leiomyosarcomas. Alternatively, thesmooth muscle tumor can be a leiomyoma. In a further embodiment, themethod further comprises contacting the smooth muscle cells with anamount effective to inhibit smooth muscle cell proliferation and/ormigration of an inhibitor of the small heat shock protein HSP27.

In a further aspect, the present invention provides a method fortreating or preventing smooth muscle spasm, comprising contacting asubject or graft in need thereof with an amount effective to inhibitsmooth muscle spasm of HSP20, or a functional equivalent thereof, suchas one or more polypeptides according to general formula I or II. In amost preferred embodiment, the one or more polypeptides arephosphorylated as disclosed above. In a further embodiment, the methodfurther comprises contacting the smooth muscle with an amount effectiveto inhibit smooth muscle cell proliferation and/or migration of aninhibitor of the small heat shock protein HSP27. In a furtherembodiment, the method further comprises contacting the smooth musclewith an amount effective of PKG to stimulate HSP20 phosphorylation, asdescribed above.

The examples below demonstrate that HSP20, and equivalents thereof, suchas the polypeptides according to general formula I and II, are effectiveat inhibiting smooth muscle spasm, such as vasospasm. While not beinglimited by a specific mechanism of action, it is believed that HSP20,and equivalents thereof, such as the polypeptides according to generalformula I and II, exert their anti-smooth muscle spasm effect bypromoting smooth muscle vasorelaxation and inhibiting contraction.

Smooth muscles are found in the walls of blood vessels, airways, thegastrointestinal tract, and the genitourinary tract. Pathologic toniccontraction of smooth muscle constitutes spasm. Many pathologicalconditions are associated with spasm of vascular smooth muscle(“vasospasm”), the smooth muscle that lines blood vessels. This cancause symptoms such as angina and ischemia (if a heart artery isinvolved), or stroke as in the case of subarachnoid hemorrhage inducedvasospasm if a brain vessel is involved. Hypertension (high bloodpressure) is caused by excessive vasoconstriction, as well asthickening, of the vessel wall, particularly in the smaller vessels ofthe circulation.

Thus, in one embodiment of this aspect of the invention, the muscle cellspasm comprises a vasospasm, and the method is used to treat or inhibitvasospasm. Preferred embodiments of the method include, but are notlimited to, methods to treat or inhibit angina, coronary vasospasm,Prinzmetal's angina (episodic focal spasm of an epicardial coronaryartery), ischemia, stroke, bradycardia, and hypertension.

In another embodiment of this aspect of the invention, smooth musclespasm is inhibited by treatment of a graft, such as a vein or arterialgraft, with HSP20, or a functional equivalent thereof, such as one ormore polypeptides according to general formula I or II, as describedabove. One of the ideal conduits for peripheral vascular and coronaryreconstruction is the greater saphenous vein. However, the surgicalmanipulation during harvest of the conduit often leads to vasospasm. Theexact etiology of vasospasm is complex and most likely multifactorial.Most investigations have suggested that vasospasm is either due toenhanced constriction or impaired relaxation of the vascular smoothmuscle in the media of the vein. Numerous vasoconstricting agents suchas endothelin-1 and thromboxane are increased during surgery and resultin vascular smooth muscle contraction. Other vasoconstrictors such asnorepinephrine, 5-hydroxytryptamine, acetylcholine, histamine,angiotensin II, and phenylephrine have been implicated in vein graftspasm. Papaverine is a smooth muscle vasodilator that has been used. Incircumstances where spasm occurs even in the presence of papaverine,surgeons use intraluminal mechanical distension to break the spasm. Thisleads to injury to the vein graft wall and subsequent intimalhyperplasia. Intimal hyperplasia is the leading cause of graft failure.

Thus, in this embodiment, the graft can be contacted with HSP20 or afunctional equivalent(s) thereof, during harvest from the graft donor,subsequent to harvest (before implantation), and/or during implantationinto the graft recipient. This can be accomplished, for example, bydelivering the recombinant expression vectors (most preferably a viralvector, such as an adenoviral vector) of the invention to the site, andtransfecting the smooth muscle cells. More preferably, delivery intosmooth muscle is accomplished by using the polypeptides of generalformula I or II that include at least one transduction domain tofacilitate entry into the smooth muscle cells. The examples belowdemonstrate the ability of the polypeptides of the invention thatcontain at least one transduction domain to be delivered into smoothmuscle cells. During graft implantation, it is preferred that thesubject receiving be treated systemically with heparin, as heparin hasbeen shown to bind to protein transduction domains and prevent them fromtransducing into cells. This approach will lead to localized proteintransduction of the graft alone, and not into peripheral tissues. Themethods of this embodiment of the invention inhibit vein graft spasmduring harvest and/or implantation of the graft, and thus improve bothshort and long term graft success.

In various other embodiments, the muscle cell spasm is associated with adisorder including, but not limited to pulmonary (lung) hypertension,asthma (bronchospasm), toxemia of pregnancy, pre-term labor,pre-eclampsia/eclampsia, Raynaud's disease or phenomenon,hemolytic-uremia, non-occlusive mesenteric ischemia (ischemia of theintestines that is caused by inadequate blood flow to the intestines),anal fissure (which is caused by persistent spasm of the internal analsphincter), achalasia (which is caused by persistent spasm of the loweresophageal sphincter), impotence (which is caused by a lack ofrelaxation of the vessels in the penis, erection requires vasodilationof the corpra cavernosal (penile) blood vessels), migraine (which iscaused by spasm of the intracranial blood vessels), ischemic muscleinjury associated with smooth muscle spasm, and vasculopathy, such astransplant vasculopathy (a reaction in the transplanted vessels which issimilar to atherosclerosis, it involves constrictive remodeling andultimately obliteration of the transplanted blood vessels, this is theleading cause of heart transplant failure).

Preferred routes of delivery for these various indications of thedifferent aspects of the invention vary. Topical administration ispreferred for methods involving treatment or inhibition of vein graftspasm, intimal hyperplasia, restenosis, prosthetic graft failure due tointimal hyperplasia, stent, stent graft failure due to intimalhyperplasia/constrictive remodeling, microvascular graft failure due tovasospasm, transplant vasculopathy, and male and female sexualdysfunction. As used herein, “topical administration” refers todelivering the polypeptide onto the surface of the organ.

Intrathecal administration, defined as delivering the polypeptide intothe cerebrospinal fluid is the preferred route of delivery for treatingor inhibiting stroke and subarachnoid hemorrhage induced vasospasm.Intraperitoneal administration, defined as delivering the polypeptideinto the peritoneal cavity, is the preferred route of delivery fortreating or inhibiting non-occlusive mesenteric ischemia. Oraladministration is the preferred route of delivery for treating orinhibiting achalasia. Intravenous administration is the preferred routeof delivery for treating or inhibiting hypertension and bradycardia.Administration via suppository is preferred for treating or inhibitinganal fissure. Aerosol delivery is preferred for treating or inhibitingasthma (ie: bronchospasm). Intrauterine administration is preferred fortreating or inhibiting pre-term labor and pre-eclampsia/eclampsia.

In practicing these various aspects of the invention, the amount ordosage range of the polypeptides or pharmaceutical compositions employedis one that effectively teats or inhibits one or more of smooth musclecell proliferation, smooth muscle cell migration, smooth muscle spasm;and/or that promotes smooth muscle relaxation. Such an inhibiting (orpromoting in the case of smooth muscle relaxation) amount of thepolypeptides that can be employed ranges generally between about 0.01μg/kg body weight and about 10 mg/kg body weight, preferably rangingbetween about 0.05 μg/kg and about 5 mg/kg body weight.

The present invention may be better understood with reference to theaccompanying examples that are intended for purposes of illustrationonly and should not be construed to limit the scope of the invention, asdefined by the claims appended hereto.

EXAMPLES Example 1

This Example illustrates a study of cyclic nucleotide-dependentphosphorylation of HSP20 in mesangial cells. The contractile phenotypeand expression of PKG is lost as smooth muscle cells are passaged inculture. Mesangial cells have been shown to maintain a contractilephenotype in culture. To determine if mesangial cells in culturecontinue to express PKG and HSP20, multiply passaged mesangial cellswere compared to multiply passaged vascular smooth muscle cells andsmooth muscle cells that had been stably transfected with PKG. The cellswere homogenized and immunoblots were performed using rabbit polyclonalantibodies against PKG and HSP20. Multiply passaged vascular smoothmuscle cells did not express PKG or HSP20. However, smooth muscle cellsthat had been stably transfected with PKG express PKG and HSP20.Cultured mesangial cells expressed similar amounts of both PKG and HSP20as the PKG transfected vascular smooth muscle cells. These data suggestthat the expression of PKG and the PKG substrate protein, HSP20, arecoordinately regulated and that the expression of these proteins may beimportant for maintaining the cells in a contractile phenotype. Sincephenotypic modulation from a contractile to a synthetic phenotype hasbeen implicated in the response to injury model of atherogenesis and inthe development of intimal hyperplasia, we propose that introducingHSP20 either via protein transduction or by gene transfection willprovide a novel therapeutic approach to maintain cells in a contractilephenotype and prevent intimal hyperplasia.

Example 2

This Example illustrates the production of the HSP20 S16A mutant whereinthe phosphorylation site (serine 16) was mutated to an alanine. The cDNAfor HSP20 was cloned into pEGFP-C2 expression vector (commerciallyavailable from Clontech, Inc.) For production of the HSP20 S16A mutant,a single nucleotide mutation was introduced in the HSP20 cDNA sequenceusing a two complimentary oligonucleotide strategy with Pfu polumerase(commercially available from Stratagene, La Jolla, Calif.). Allsequences were confirmed for orientation, the presence of theappropriate mutations, and the absence of other mutations, using a 377Perkin-Elmer ABI Prism DNA sequencer (Foster City, Calif.). Similartechniques can be used to mutate the serine 16 for an aspartic orglutamic acid.

Example 3

This experiment illustrates that genetic manipulation of muscle likecells can alter their ability to contract. Specifically, engineering thecells to overexpress HSP20 prevents them from contracting (going into astate of spasm). If the cells overexpress a mutated form of HSP20 thatcannot be phosphorylated, they remain contracted (in spasm) even whentreated with potent agents which cause relaxation. This experimentdemonstrates that the phosphorylation of HSP20 is the seminal eventrequired for muscles to relax.

The experiment was performed in the following fashion: Mesangial cellswere transfected with vectors containing green fluorescent protein (GFP)alone, GFP fused to the 5′ end of the wild type cDNA for HSP20 (WT), orGFP fused to an HSP20 construct in which the PKA phosphorylation sitewas mutated to an alanine (S16A-HSP20) (MUT). The cells were plated on asilicone rubber substrata in the presence of serum for 48 hours. Theplates were then placed on the stage of a Zeiss Inverted FluorescenceMicroscope with DeltaVision image acquisition and deconvolution software(Applied Precision, Issaquah, Wash.). The DeltaVision software wasconfigured to eight different cells (x and y axis) on each plate with 7z-axis images taken at 2 nm intervals. Fluorescent and phase contrastimages were obtained such that the cells and silicone membrane wereimaged in close succession. During the scanning process, the z-axis ofeach cell was monitored to assure that the imaging stacks weremaintained at the appropriate level as the cells relaxed on the siliconemembrane. Baseline images were acquired for one hour. The cells werethen treated with the cells were treated with dibutyryl cAMP (10 ΦM) for0 minutes, 30 minutes, 60 minutes, or 90 minutes, and the results areillustrated in FIG. 1.

The control cells expressing the green fluorescent protein (GFP) inwhich the HSP20 was not changed relaxed over time (the wrinkles underthe cells disappeared) when treated with dibutyryl cAMP. The cells overexpressing HSP20 tagged to GFP (WT) did not form wrinkles; they wereunable to contract or go into spasm. The cells expressing the mutatedform of HSP20 that could not be phosphorylated (S16A-HSP20) (MUT) formedabundant wrinkles (contracted) but did not relax (remained in spasm) inresponse to dibutyryl cAMP. This figure is representative of 6 separatetransfections in which at least 12 cells were imaged with each constructand the aggregate data is illustrated graphically (*=p<0.05 compared tothe initial number of wrinkles). Similar findings were observed whencyclic nucleotide-dependent signaling pathways were activated withsodium nitroprusside (10 μM), dibutyryl cGMP (10 μM), or forskolin (10μM) (data not shown). There were no changes in the wrinkles in thesubstrata in untreated cells imaged for 90 minutes (data not shown).These data demonstrate that over expression of wild type HSP20 inhibitscontraction of the cells and expression of the S16A-HSP20 mutatedprotein inhibits relaxation. Thus, these data show that thephosphorylation of HSP20 is necessary and sufficient for the relaxationof smooth muscles, and suggests that phosphorylation of HSP20 representsa point in which the cyclic nucleotide signaling pathways converge toprevent contraction or cause relaxation.

Example 4

This experiment demonstrates that transduction of peptide analogues ofphosphorylated HSP20 relaxes muscle-like cells. Phosphopeptide analoguesof HSP20 were synthesized containing the TAT sequence(NH₂-βAGGGGYGRKKRRQRRRWLRRAS*APLPGLK-COOH) (referred to asFITC-TAT-HSP20) (SEQ ID NO:304) (the asterisk indicates that the “S”residue is phosphorylated). Mesangial cells were plated on a siliconesubstrata in the presence of serum and after 48 hours the cells weretreated with the FITC-TAT-HSP20 phosphopeptide (50 μM). The number ofwrinkles under the cells was determined at the time points indicatedusing phase contrast microscopy (n=10, *=p<0.05 compared to time 0).Results of this Experiment are illustrated in FIG. 2. Treatment of thecells with the phosphopeptide analogue of HSP20 led to a time dependentloss of wrinkles (relaxation of the cells). This experiment demonstratesthat transduction of phosphopeptide analogues of HSP20 also relaxes thecells.

Example 5

This experiment demonstrates that transduction of phosphopeptideanalogues of HSP20 relax and prevent spasm in intact strips of vascularsmooth muscle. Transverse strips of bovine carotid artery smooth muscle,denuded of endothelium, were suspended in a muscle bath containingbicarbonate buffer (120 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO₄, 1.0 mMNaH₂PO₄, 10 mM glucose, 1.5 mM CaCl₂, and 25 mM Na₂HCO₃, pH 7.4),equilibrated with 95% O₂/5% CO₂, at 37° C. at one gram of tension for 2hours. The muscles were pre-contracted with serotonin (1 μM for 10minutes) and cumulative doses of FITC-phosphoHSP20-TAT, FITC-scrambledphosphoHSP20-TAT (FITC-NH₂-βAGGGGYGRKKRRQRRRPRKS*LWALGRPLA-COOH, opencircles) (SEQ ID NO:305), or FITC-TAT (FITC-NH₂-βAGGGGYGRKKRRQRRR,closed triangles) (SEQ ID NO:306) were added every 10 minutes. The forceis depicted as a percentage of the maximal serotonin contraction (n=5,*=p<0.05 compared to 0 peptide added). Representative strips were fixedin 4% paraformaldehyde and examined under fluorescence microscopy (40×magnification). The internal elastic lamina (IEL) autofluoresced, andthe media and adventitia (ADV) also displayed fluorescence(not shown).

The results of this Experiment are illustrated in FIG. 3. Transductionof pre-contracted strips of intact bovine corotid artery smooth musclewith the FITC-TAT-HSP20 phosphopeptide led to a dose dependent decreasein serotonin pre-contracted muscles. Peptides containing the scrambledsequence or FITC-TAT alone had no significant effect on contractileforce. This shows that the phosphopeptide analogues relax and preventspasm in intact vascular smooth muscles. There was a diffusefluorescence staining pattern of the muscle strips after transductionwith the FITC-TAT-HSP20 phosphopeptide which shows that the peptidesenter the muscles.

Example 6

This experiment shows that a different transducing peptide can introducethe HSP20 phosphopeptide analogues. In addition, it demonstrates thatphosphopeptide analogues of HSP20 relax and prevent spasm in smoothmuscles from a different vascular bed in a different species. Finally,it shows that transduction of HSP20 analogues relax and prevent spasm inmuscles in which an intact endothelium is present.

Rings of porcine coronary artery in which the endothelium was notdenuded, were suspended in a muscle bath containing bicarbonate buffer(120 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO₄, 1.0 mM NaH₂PO₄, 10 mM glucose,1.5 mM CaCl₂, and 25 mM Na₂HCO₃, pH 7.4), equilibrated with 95% O₂/5%CO2, at 37° C. at one gram of tension for 2 hours. The muscles werepre-contracted with serotonin (1 μM for 10 minutes) and cumulative dosesof PTD-pHSP20 (NH₂-βAYARRAAARQARAWLRRAS*APLPGLK-COOH, closed circles)(SEQ ID NO:307) or PTD-scrambled-pHSP20(NH₂-βAYARRAAARQARAPRKS*LWALGRPLA-COOH open circles) (SEQ ID NO:308)were added every 10 minutes (FIG. 4). The percentage of relaxation isdepicted as a percentage of the maximal serotonin contraction (n=5,*=p<0.05 compared to 0 peptide added). The concentrations of peptideused are depicted on the x axis. Representative rings were treated withpeptides (1 mM final concentration) linked to FITC (15 minutes at 37°C.), fixed in 4% paraformaldehyde and examined under fluorescencemicroscopy (40× magnification).

The results of this experiment are illustrated in FIG. 4. This showsthat phosphopeptide analogues of HSP20 relax and prevent spasm inmuscles from a different species and different vascular bed. There wasmarked fluorescence in the strips treated with protein transductionanalogues. This demonstrates that a protein transduction peptide that isdifferent than TAT can transduce the phosphopeptide analogue and relaxand prevent spasm in muscles.

Example 7

This experiment shows that protein transduction of phosphopeptideanalogues of HSP20 can relax and prevent spasm in non-vascular smoothmuscles.

The internal anal sphincter was obtained from a human pathology specimenafter an abdominal perineal resection for cancer. The smooth muscleswere equilibrated in a muscle bath as described in example 6. Themuscles developed tonic sustained contractions when warmed in thebicarbonate buffer. These contractions relaxed with the addition of theguanylate cyclase activator, sodium nitroprusside (SNP). When the sodiumnitroprusside was washed out of the bath, the muscles again contractedspontaneously. The muscles were then treated with the PTD-phosphopeptideanalogues of HSP₂-(NH₂-βAYARRAAARQARAWLRRAS*APLPGLK-COOH, 1 mM finalconcentration) (SEQ ID NO:307). The muscles relaxed and spasm wasprevented in response to the phosphopeptide analogues and the relaxationwas sustained.

Intestinal smooth muscle was obtained from the tinea coli of a pig.These muscles were equilibrated in a muscle bath as described in example6. The muscles produced transient contractions in response to highextracellular potassium chloride (KCl 110 mM) and in response tocarbachol (10⁻⁶ M). The muscles were then treated with a dextran gelcontaining the PTD-phosphopeptide analogues of HSP20(NH₂-βAYARRAAARQARAWLRRAS*APLPGLK-COOH, 1 mM final concentration) (SEQID NO:307) and treated with carbachol. Treatment with the phosphopeptideanalogues significantly attenuated the contractile response tocarbachol.

Tracheal and corpra cavernosal smooth muscles were obtained from a NewZealand White rabbits after sacrifice. The muscles were equilibrated ina muscle bath as described in example 6. The tracheal muscles werepre-contracted with carbachol and the corpra cavernosal smooth muscleswere pre-contracted with norepinephrine. The muscles were then treatedwith the PTD-phosphopeptide analogues of HSP20(NH2-βAYARRAAARQARAWLRRAS*APLPGLK-COOH) (SEQ ID NO:307). Both thetracheal and the corpra cavernosal muscles relaxed and spasm wasprevented in response to the phosphopeptide analogues.

The results of these experiments show that the phosphopeptide analoguesof HSP20 relax and prevent spasm in human anal sphincter smooth muscles,porcine intestinal smooth muscle, rabbit tracheal smooth muscles, andrabbit corpra cavernosal smooth muscles.

Example 8

This experiment shows that protein transduction of phosphopeptideanalogues of HSP20 can relax and prevent spasm in human saphenous veinsmooth muscles.

Human saphenous vein was obtained from remnants that were discardedafter vascular bypass operations. Rings of the saphenous vein wereequilibrated in a muscle bath as described in experiment 4. The ringswere treated with a 7.5% dextran gel alone, or a 7.5% dextran gelcontaining the phosphopeptide analogues of HSP20(NH₂-βAYARRAAARQARAWLRRAS*APLPGLK-COOH) (SEQ ID NO:307). The rings werethen treated with serotonin (1 uM). The rings treated with the 7.5%dextran gel alone contracted in response to serotonin. However, therings treated with the 7.5% dextran gel that contained the HSP20phosphopeptide analogues did not contract in response to serotonin.

These results show that the phosphopeptide analogues of HSP20 preventspasm (contraction) of human saphenous vein smooth muscles.

Example 9

This experiment was performed to demonstrate that smaller peptideanalogues of phosphorylated HSP20 relax and prevent spasm of smoothmuscles even more effectively than the larger analogues.

Rings of rabbit aorta in which the endothelium was not denuded, weresuspended in a muscle bath containing bicarbonate buffer (120 mM NaCl,4.7 mM KCl, 1.0 mM MgSO₄, 1.0 mM NaH₂PO₄, 10 mM glucose, 1.5 mM CaCl₂,and 25 mM Na₂HCO₃, pH 7.4), equilibrated with 95% O₂/5% CO₂, at 37° C.at one gram of tension for 2 hours. The rings were pre-contracted withnorepinephrine (10⁻⁷ M) and treated with RRRRRRApSAPLP (SEQ ID NO:309)or RRRRWLRRApSAPLP (SEQ ID NO:310). Both peptides caused rapid andcomplete relaxation and inhibition of spasm of the muscles, and therelaxation was faster and the muscles remained in a relaxed state forlonger than when the longer peptides were used. The peptides used wereprepared by Fmoc-based peptide synthesis, and the peptides retained theFmoc moiety at the amino terminus of the peptide.

These data show that poly arginine sequences can transduce the HSP20analogues and induce relaxation and that the smaller sequence ApSAPLP(SEQ ID NO:3) (where “p’ indicates that the S residue is phosphorylated)causes rapid and complete relaxation and inhibition of spasm.

Example 10

This experiment illustrates that HSP20 is expressed in mesangial cellsand in rat aortic smooth muscle cells that have been stably transfectedwith PKG. It also illustrates that relaxation of mesangial cells isassociated with increases in the phosphorylation of HSP20.

Homogenates of mesangial cells (FIG. 5, lane 1), rat aortic smoothmuscle cells (FIG. 5, lane 2), and PKG transfected rat aortic smoothmuscle cells (FIG. 5, lane 3) were immunoblotted for PKG (FIG. 5, panelA) or HSP20 (FIG. 5, panel B). In a separate experiment, mesangial cellswere untreated (FIG. 5, panel C) or treated with dibutyryl cAMP (10 μM,15 minutes, FIG. 5, panel D). The proteins were separated by2-dimensional electrophoresis, transferred to immobilon and probed withanti-HSP20 antibodies. Increases in the phosphorylation of HSP20 lead toa shift in the electrophoretic mobility of the protein to a more acidicisoform (arrow).

These data show that activation of cyclic nucleotide-dependent signalingpathways in mesangial cells (as shown in FIG. 2) is associated withphosphorylation of HSP20.

Example 11

This experiment illustrates that cells expressing HSP20 (stably PKGtransfected cells) are able to contract.

Rat aortic smooth muscle cells that are multiply passaged or that stablyexpress PKG were cultured on a silicone substrata in the presence ofserum. The cells were imaged with phase contrast microscopy(10×magnification). The multiply passaged cells did not form wrinkles onthe substrata whereas the PKG transfected cells formed wrinkles. Todetermine if the wrinkles were reversible, PKG transfected cells weretreated with dibutyryl cAMP (10 uM) for 30 minutes. Dibutyryl cAMP ledto a decrease in wrinkle formation.

Taken together with example 10, these results show that the expressionof HSP20 is associated with a contractile phenotype. Vascular smoothmuscle cells exist in widely divergent phenotypes. In the normal vesselwall, the smooth muscle cells are in a well differentiated contractilephenotype and are capable of generating force. In response to injury, orcell culture conditions, the cells modulate to a synthetic or secretoryphenotype. These cells proliferate and secrete matrix proteinscontributing to intimal hyperplasia. Phenotypic modulation is associatedwith changes in gene expression, protein expression, morphology, andphysiologic responses. This leads to pathologic narrowing of the vessellumen which occurs in atherosclerosis and intimal hyperplasia. Thisleads to stenotic lesions and ultimately occlusion of the vessel. Thus,maintaining expression of HSP20 is important for the maintenance of thecontractile phenotype.

Example 12

Cellular processes such as cell adhesion, cytokinesis, cell motility,migration, and contraction all require dynamic reorganization of theactin cytoskeleton These experiments show that the phosphorylation ofHSP20 modulates changes in these actin filaments.

Transfected mesangial cells were fixed and the actin filaments werestained with fluorescent-labeled phalloidin. Mesangial cells weretransfected with EGFP alone (EGFP), S16A-HSP20 (MUT-EGFP, or wild typeHSP20 (WT-EGFP). The cells were plated on a glass slides, and nottreated (CONT) or treated with dibutyryl cAMP (10 μM, for 30 minutes,db-cAMP). The cells were fixed and stained with rhodamine phalloidin.Dibutyryl cAMP led to a loss of central actin stress fibers in EGFP butnot S 16A-HSP20 cells. In the cells overexpressing HSP20 the actinfibers were peripherally localized. The results of this experiment areillustrated in FIG. 6.

These experiments show that activation of cyclic nucleotide-dependentsignaling pathways, which lead to increases in the phosphorylation ofHSP20, are associated with a loss of central actin stress fibers.Over-expression of HSP20 was also associated with a loss of actin stressfibers. In cells overexpressing a mutated form of HSP20 in which theserine has been replaced with an alanine (S16A-HSP20) and cannot bephosphorylated, there is no loss of these central actin fibers withactivation of cyclic nucleotide-dependent signaling pathways. Thesestudies demonstrate that phosphorylation of HSP20 is associated withchanges in actin fiber formation.

Example 13

This experiment shows that protein transduction of smooth muscle cellswith phosphopeptide analogues of HSP20 also leads to changes in actinfiber formation.

Rat aortic smooth muscle cells were treated with lyophosphatidic acid(LPA) in the presence and absence of FITC-TAT-pHSP20(FITC-NH₂-βAGGGGYGRKKRRQRRRWLRRAS*APLPGLK-COOH, 50 uM) (SEQ ID NO:307)Lysophosphatidic acid (LPA) is a substance which promotes actin fiberformation. Inhibition of the actions of LPA have been shown to inhibitintimal hyperplasia.

The cells were fixed and stained with phalloidin and images wereobtained with confocal microscopy. LPA led to robust actin stress fiberformation, whereas there was a loss of central actin stress fibers inthe cells treated with LPA in the presence of the FITC-TAT-pHSP20peptide. These studies show that protein transduction with thephosphopeptide analogues of HSP20 inhibit LPA-induced actin fiberformation. These studies confirm that HSP20 has a direct role inmodulating actin fiber formation.

Example 14

Cell adhesion formation involves the interaction between integrins andextracellular matrix substrates. This induces integrin clustering. Thecells then form actin microfilaments and the cells spread. If theappropriate signals are provided by the matrix, the cells proceed toorganize their cytoskeleton as characterized by the formation of focaladhesions and actin-containing stress fibers. Cell adhesion is a dynamicreversible process integral to cell migration. Activation of cGMP leadsto focal adhesion disassembly. These studies show that phosphopeptideanalogues of HSP20 mediate focal adhesion disassembly.

Bovine aortic endothelial cells were plated on glass coverslips(80K-100K cells) in DMEM plus 10% FBS over night (24 wells plate). Thecells were serum starved (no serum) for one hour and incubated in thepresence of the peptide analogues of HSP20[NH₂-βAYARRAAARQARAWLRRAS*APLPGLK-COOH-pHSP20 (10 uM) (SEQ ID NO:307) orscrambled analogues of HSP20[NH₂-βAYARRAAARQARAPRKS*LWALGRPLA-COOH-scHSP20 (10 uM)] (SEQ ID NO:308)for 30 minutes. The cells were fixed with 3% glutaraldehyde and thenumber of focal adhesions was detected with interference reflectionmicroscopy. The Hep I peptide was used as a positive control. Thispeptide binds to the heparin binding site of thrombospondin in inducesfocal adhesion disassembly. Both Hep I and pHSP20 led to focal adhesiondisassembly. The results are illustrated in FIG. 7.

Treatment with PTD-pHSP20 led to a disassociation of focal adhesionssimilar to the disassociation induced by a peptide from theamino-terminal heparin-binding domain of thrombospondin 1 (Hep 1). Thescrambled peptide had no significant effect on focal adhesiondisassembly. These studies show that phosphorylated HSP20 mediates focaladhesion disassembly. This weakens cell attachment and prevents theformation of the attachments necessary for cell migration.

Example 15

This Experiment shows that the phosphorylated peptide analogues of HSP20directly inhibit cell migration.

Confluent A10 cells were serum starved (0.5% fetal bovine serum, FBS)for 48 hours. A linear wound was made in the smooth muscle cellmonolayer using a rubber scraper and the scratched edges were markedusing metal pins. The cells were changed to 10% FBS media containingPTD-pHSP20 (NH2-βAYARRAAARQARAWLRRAS*APLPGLK-COOH (SEQ ID NO:307), orPTD-scrambled-pHSP20 (NH₂-βAYARRAAARQARAPRKS*LWALGRPLA-COOH (SEQ IDNO:308) (50 μM) and incubated for 24 hours. The cells were fixed andstained with hematoxylin. The number of cells migrating into a 1 cm²scratched area were counted as an index for migration. In additionalexperiments, the migration of A10 cells was determined in a Boydenchamber assay. In both cases the phosphopeptide analogue of HSP20 led toinhibition of migration. FIG. 8 illustrates the results of theseexperiments.

These results show that transduction of phosphopeptide analogues ofHSP20 inhibits serum-induced migration of smooth muscle cells.

Example 16

This experiment shows that transduction of phosphopeptide analogues ofHSP20 inhibits serum-induced proliferation of smooth muscle cells.

A10 cells were serum starved for 3 days. The cells were then treatedwith media containing 10% fetal bovine serum, PTD-pHSP20(NH₂-βAYARRAAARQARAWLRRAS*APLPGLK-COOH (SEQ ID NO:307), orPTD-scrambled-pHSP20 (NH₂-βAYARRAAARQARAPRKS*LWALGRPLA-COOH (SEQ IDNO:308) (50 μM). After 24 hours cell counts were performed. The numberof cells per well in the serum starved plates averaged 109 cells/well(+/−7.4) compared to 276+/−6.1 in wells containing 10% fetal bovineserum (FBS). In the presence of FBS, the phosphopeptide analogues ofHSP20 containing a transduction domain inhibited of smooth muscleproliferation, 149+/−14.6 compared to transduction of the scrambledphosphopeptide analogue of HSP20 (242.3+/−15.3 cells/well). The resultsof these experiments are illustrated in FIG. 9.

The results from these examples demonstrate that HSP20 is associatedwith a contractile phenotype and that transduction of phosphopeptideanalogues of HSP20 inhibit actin fiber formation, focal adhesionformation, smooth muscle cell migration and smooth muscle proliferation.These are cellular processes that lead to intimal hyperplasia, and otherdisorders as discussed throughout the application.

Example 17

These experiments show that HSP20 inhibits intimal hyperplasia in humansaphenous vein grafts.

Segments of human saphenous vein were cultured in media containing 30%fetal bovine serum. The segments were treated for 14 days with mediacontaining serum alone, serum and the phosphopeptide analogue of HSP20(PTD-pHSP20 (NH₂-βAYARRAAARQARAWLRRAS*APLPGLK-COOH, 10 :M) (SEQ IDNO:307), or the PTD-scrambled phosphopeptide analogue(NH₂-βAYARRAAARQARAPRKS*LWALGRPLA-COOH, 10 : μM). (SEQ ID NO:308) Therings were fixed in formalin, stained with hematoxylin and eosin, andthe intimal area was measured morphometrically. There was a significantreduction in intimal area in the rings transduced with thephosphopeptide analogues of HSP20 compared to the rings treated withserum alone or serum and transduction of the scrambled analogue.

These results show that intimal hyperplasia can be inhibited in humansaphenous vein segments by transduction of the phosphopeptide analoguesof HSP20.

Example 18

This Experiment illustrates that plant cells can be engineered toproduce recombinant HSP20.

Tobacco BY-2 cells were transformed with vector alone (vectortransformed) or with His tagged HSP20 constructs (6Xhis-HSP20transformed). Western blots were performed on cells lysates usinganti-6Xhis monoclonal antibodies. There is immunoreactivity of a 20 kDapolypeptide in the HSP20 lysates but not the empty vector transformedlysates.

Optical sections from confocal immunofluorescence images of processedtobacco cells transiently-expressing either myc-epitope tagged HSP20,probed with anti-myc antibodies, HSP20, probed with anti-HSP20antibodies, TAT-HSP20, probed with anti-HSP20 antibodies, orHISTAT-HSP20, probed with anti-his antibodies. Expression is presentwith all 4 constructs (bar=100 μm).

These data show that plants can be engineered to produce proteins thatcontain a protein transduction sequence and the HSP20 molecule. Thisrepresents an alternative source of production of HSP20.

1. A polypeptide consisting of a sequence according to general formulaI:X1-X2-[X3-A(X4)APLP-X5-]_(u)-X6 wherein X1 is absent or is one or moremolecules comprising one or more aromatic ring; X2 is absent orcomprises a transduction domain; X3 is 0, 1, 2, 3, or 4 amino acids ofthe sequence WLRR (SEQ ID NO:1); X4 is selected from the groupconsisting of S, T, Y, D, E, phosphoserine analogs and phosphotyrosineanalogs; X5 is 0, 1, 2,or 3 amino acids of a sequence of genus Z1-Z2-Z3,wherein Z1 is selected from the group consisting of G and D; Z2 isselected from the group consisting of L and K; and Z3 is selected fromthe group consisting of S and T; and X6 is absent or comprises atransduction domain; and wherein u is 1-5.
 2. The polypeptide of claim 1wherein either X2 or X6 comprises a transduction domain.
 3. Thepolypeptide of claim 1 wherein X4 is phosphorylated.
 4. The polypeptideof claim 1 wherein X1 is a molecule comprising an aromatic ring.
 5. Thepolypeptide of claim 4 wherein X1 is selected from the group consistingof F, Y, W; and compounds comprising 9-fluroenylmethyl.
 6. A polypeptidecomprising a sequence according to general formula II:X1-X2-[X3-A(X4)APLP-X5-]_(u)-X6 wherein X1 is absent or is one or moremolecules comprising one or more aromatic ring; X2 is absent orcomprises a cell transduction domain; X3 is 0-14 amino acids of thesequence of heat shock protein 20 between residues 1 and 14 of SEQ IDNO:297; X4 is selected from the group consisting of S, T, Y, D, E,phosphoserine analogs and phosphotyrosine analogs; X5 is 0-140 aminoacids of heat shock protein 20 between residues 21 and 160 of SEQ IDNO:297; X6 is absent or comprises a cell transduction domain; andwherein at least one of X2 and X6 comprise a transduction domain.
 7. Thepolypeptide of claim 5 wherein X4 is phosphorylated.
 8. The polypeptideof claim 5 wherein X1 is a molecule comprising an aromatic ring.
 9. Thepolypeptide of claim 8 wherein X1 is selected from the group consistingof F, Y, W; and compounds comprising 9-fluroenylmethyl.
 10. Apharmaceutical composition, comprising one or more polypeptidesaccording to claim 1, and a pharmaceutically acceptable carrier.
 11. Apharmaceutical composition, comprising one or more polypeptidesaccording to claim 5, and a pharmaceutically acceptable carrier.
 12. Anisolated nucleic acid sequence encoding the polypeptide of claim
 1. 13.An isolated nucleic acid sequence encoding the polypeptide of claim 5.14. An expression vector comprising the nucleic acid of claim
 12. 15. Anexpression vector comprising the nucleic acid of claim
 13. 16. A hostcell comprising the expression vector of claim
 14. 17. A host cellcomprising the expression vector of claim
 15. 18. An improved biomedicaldevice, wherein the biomedical device comprises one or more polypeptidesaccording to claim 1 disposed on or in the biomedical device.
 19. Animproved biomedical device, wherein the biomedical device comprises oneor more polypeptides according to claim 5 disposed on or in thebiomedical device.
 20. (canceled)
 21. (canceled)
 22. (canceled) 23.(canceled)
 24. (canceled)
 25. (canceled)
 26. (canceled)
 27. (canceled)28. (canceled)
 29. A composition comprising: (a) a polypeptide accordingto claim 1; and (b) an inhibitor of HSP27.
 30. A composition comprising:(a) a polypeptide according to claim 5; and (b) an inhibitor of HSP27.31. (canceled)
 32. (canceled)
 33. (canceled)
 34. A polypeptidecomprising a sequence according to the formula X1-X2-SEQ ID NO:300-X6,wherein X1 is absent or is one or more molecules comprising one or morearomatic ring; X2 is absent or comprises a cell transduction domain; andX6 is absent or comprises a cell transduction domain; and wherein atleast one of X2 and X6 comprise a transduction domain.
 35. Apharmaceutical composition comprising the polypeptide of claim 34 and apharmaceutically acceptable carrier.